3 Methods
3 METHODS
3.1 General DNA techniques
3.1.1 Spectrophotometric quantitation of nucleic acid
(Sambrook et al., 1989, page E.5)
If the sample is pure (without significant amounts of contaminants as proteins, phenol or
agarose) spectrophotometric quantitation of the DNA is reliable. The nucleic acids in both DNA
and RNA absorbs light in the ultraviolet range (200-400 nm), with an absorption peak at 260 nm.
Proteins have an absorption peak at 280 nm. Spectrofotometric readings should be taken at both
wavelengths and Kalckar's formula (the OD260/OD280 ratio) should be used to provide an
estimate for the purity of the nucleic acid.
Pure preparations of DNA or RNA have the OD260/OD280 values of 1.8 and 2.0 respectively. If
there is contamination with proteins, the ratio will be significantly less than the values given
above. The concentration of DNA or RNA can be estimated by Beer-Lamberts law.
A=cl
A = absorbance
 = extinction coefficient
c = concentration of sample
l = length of light pathway in cm
An OD260 nm measurement of 1.0 with l= 1 cm corresponds to approximately
50 g/ml double stranded DNA
40 g/ml single stranded DNA or RNA
20 g/ml single stranded oligonucleotides
Protocol
1. Dilute the sample in dH2O and measure the absorbance at 260 and 280 nm in a
spectrophotometer.
Use quartz cuvettes when measuring, since plastic absorbs light in the ultraviolet range. Use dH 2O to
calibrate the spectrophotometer.
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3.1.2 Digestion of DNA with restriction enzymes
(Sambrook et al., 1989, page 5.31-32).
Restriction enzymes recognise specific, often palindromic, sequences in double stranded DNA,
and cleave these by hydrolysis of the phosphodiester bonds in DNA. These enzymes are
classified into three groups, type I, type II and type III. Neither type I nor type III are widely used
in molecular cloning.
Typical type II restriction enzymes recognise specific DNA sequences that are four, five or six
nucleotides in length. The location of cleavage sites within a sequence differs from enzyme to
enzyme. Some cleave both strands exactly in the middle of the sequence, creating fragments with
blunt ends. Others cleave at similar locations some basepairs apart on opposite strands in the
DNA, creating DNA fragments with single stranded termini.
Protocol
The following procedure is designed for a typical single stranded reaction, containing 0.2 – 5 g
DNA. For digestion of larger amounts of DNA, the reaction mixture should be scaled
appropriately. It is important to optimise the temperature, incubation time, pH and salt
concentration for optimal digestion of DNA. Use buffer, incubation time and temperature
recommended by the manufacturer.
1. Transfer the DNA into a sterile eppendorf tube and mix with dH2O and restriction buffer.
Then add restriction enzyme(s). A typical reaction contains.
1-5
1-2
2
20 – X
l vector (2-5 g DNA)
l restriction enzyme ( 1 U/g DNA)
l 10 x restriction buffer (recommend by manufacturer)
l ddH2O (X = total volume of other solutions).
One unit restriction enzyme is defined as the enzyme concentration needed for cutting of 1 g DNA
at 37 C in one hour. It is recommended to use 2 U/g DNA for small scale preparations and
plasmides. In large scale preparations less than 1 U /g DNA could be used if the incubation time is
increased. More than one restriction enzyme can be used simultaneously, but make sure that the
restriction enzyme(s) contribute less than 1/10 of the volume in the final reaction mixture. Restriction
enzymes are stored in glycerol, which can inhibit the enzyme activity by making the solution viscous.
2. Incubate the reaction mixture for the time and at the temperature required for optimal
cutting by the restriction enzyme, usually 1 hour at 37 C.
3. Analyse the digestion reaction by agarose gel electrophoresis (page 40).
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3 Methods
3.1.3 Ligation of DNA
(Sambrook et al., 1989, page 5.61-71)
Ligation of DNA is catalysed by DNA ligase, an enzyme that joins two pieces of DNA. The
bacteriophage T4 DNA ligase catalyses the formation of phosphodiester bonds between adjacent
3´-hydroxyl and 5´-phosphate termini in DNA. The enzyme catalyses ligation of DNA fragments
with both blunt and single stranded termini ends (Figure 3-1).
P-
A A T T C
HO-G
G-OH
C T T A A-P
Annealing
G A A T T C
C T T A A G
DNA Ligase
seals the nicks
G A A T T C
C T T A A G
Figure 3-1: Ligation of EcoRI restriction site.
The single stranded termini ends anneal and DNA ligase catalyses the formation of phosphodiester bonds between
adjacent 3´-hydroxyl and 5´-phosphate termini in DNA.
The reaction is performed in eppendorf tubes with small volumes (10-15 l) facilitating
annealing of two DNA fragments with compatible termini. After annealing, the DNA ligase seals
the single stand nick in the DNA. This protocol is optimised for single strand termini ligation
reactions.
Protocol
1. Use the formula below to estimate the amount of the vector and insert needed for optimal
ligation.
X ng insert 
(Y bp insert)  (50 ng vector)
(total bp in vector)
It is recommended to use reactants from
a 1:1 up to 1:3 vector:insert ratio.
2. Mix vector and insert in an eppendorf tube.
3. Add to final concentrations
1x ligation buffer (recommended by the manufacture)
0,25 mM ATP
1,5 U bacteriophage T4 DNA ligase
4. Incubate the reaction mixture at 16 C until next day.
Optimal ligation temperature for bacteriophage T4 DNA ligase is 37 C, but this temperature does not
facilitate annealing of complementary DNA fragments.
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3 Methods
3.1.4 Separation of DNA by agarose gel electrophoresis
(Sambrook et al., 1989, page 6.3)
The mobility of a molecule in an electric field depends on the charge, size and structure/shape of
the molecule, the electric field and the pore size in the supporting matrix. In agarose
electrophoresis, the supporting matrix is agarose, and the method separates DNA molecules by
size. Large molecules migrate more slowly because of greater frictional drag, since they have
greater difficulties migrating through the pores in the gel than smaller molecules. The separation
is inversely proportional to the log10 of the number of base pairs in the DNA molecule. At
physiological pH DNA is negatively charged and migrates towards the anode (Figure 3-2).
The DNA molecules are visualised by ethidium bromide, usually added to the gel before it sets.
The ethidium bromide intercalates between the bases in a double stranded DNA, and the
complex is fluorescent when exposed to ultraviolet light. Detection of as little as 1-10 ng of DNA
is possible. During electrophoresis, ethidium bromide migrates towards the cathode (opposite of
DNA). Extended electrophoresis can remove much of the ethidium bromide from the gel, making
detection of small fragments difficult. The binding of ethidium bromide to DNA during
electrophoresis reduces the mobility of the DNA molecules by approximately 15 %.
Wells for loading
of samples
Cathode
Anode
-
+
Migration of DNA
Figure 3-2: Agarose gel electrophoresis
During agarose electrophoresis, the DNA migrates towards the anode in an electric field.
Reagents
Sample buffer (10 x)
50 % glycerol
50 mM EDTA
0,25 % (w/v) bromophenol blue
TAE buffer (50 x)
2 M Tris base
242 g
glacial acetic acid
57,1 ml
0,05 mM EDTA (pH 8.0)
40
Ethidium bromide (stock solution)
Dilute 10 mg ethidium bromide per ml dH2O.
3 Methods
Protocol
1. Seal the open ends of a clean, dry, plastic tray with tape to form a mold. Set the mold
horizontal on the table.
2. Prepare sufficient electrophoresis buffer (1 x TAE buffer). (It is recommended to use the
same buffer in both the gel and the running buffer). Add powdered agarose in an aliquot
of the buffer and melt it in a microwave oven. (For a small 1 % agarose gel, mix 0.5 g
agarose in 50 ml TAE-buffer.)
3. Cool the solution to 50-60 C, and add ethidium bromide to 1 g/ml final concentration in
gel.
4. Position the comb 0,5 to 1 mm above the plate in position close to the cathode, and pour
the warm agarose solution into the mold. Remove air bubbles. Let the gel set for 30-45
minutes.
5. Carefully remove the comb and the tape and mount the gel in the electrophoresis
apparatus. Add 1 x TAE buffer to cover the gel with a depth of about 1 mm. Wash the
wells with 1 x TAE buffer.
6. Mix the DNA with the sample buffer, and slowly load the mixture into the well.
7. Attach the electric leads and apply voltage. Run the electrophoresis at 3-10 V/cm (50-150
V in small gel chamber) for 1 to 3 hours.
8. Detect the DNA fragment with an UV detector or in a fluorImager. Restain the gel in
buffer containing 0,5 g/ml ethidium bromide, until satisfactory staining.
3.1.5 WizardTM PCR Preps DNA Purification System
Agarose electrophoresis (page 40) is often used to separate DNA fragments before purification,
but the agarose must be removed to enable further downstream enzymatic reactions. Thus, after
the agarose electrophoresis, the DNA is separated form the agarose by the WizardTM PCR preps
DNA purification Systems. Linear or circular DNA with fragment size from 200 – 5000 is
immobilised on an anionic resin, then washed in several steps and in the end eluted.
Protocol
Protocol as described in the “WizardTM PCR Preps DNA purification Systems for Rapid
Purification of DNA Fragments”.
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3 Methods
3.1.6 Polymerase chain reaction (PCR)
(Sambrook et al., 1989, page 14.2-14.5)
The polymerase chain reaction (PCR) is used to amplify segment(s) of DNA that are situated
between two regions of known sequence. Two short oligonucleotides are used as primers for a
series of synthetic reactions that are catalysed by a DNA polymerase. The two primers are
complementary (to opposite DNA strands) to the two known sequences at the end of the
segment(s) to be amplified (illustration beneath).
Primer 1
5'
DNA segment to be amplified
3'
3'
5'
3'
5'
5'
3'
Primer 2
The template DNA is first denatured by heating in the presence of a large molar excess of each of
the two primes and the four dNTPs. The reaction mixture is then cooled to a temperature that
allows the oligonucleotide primers to anneal to their target sequence. After annealing, the primers
are extended in a reaction catalysed by the DNA polymerase, which syntheses the
complementary strand. The cycle of denaturation, annealing, and DNA synthesis is then repeated
many times. The major product of the reaction is a segment of double stranded DNA, whose
termini is defined by the 5'-termini of the oligonucleotide primers, and whose length is defined
by the distance between the two primers. Twenty cycles of PCR amplification, increases the
amount of the target sequence around one-million fold with high specificity.
The extreme heating to denature DNA and facilitate proper annealing of primers and single
strand DNA, also inactive most enzymes. Thus a heat stabile polymerase (Taq polymerase) is
used to catalyse the reaction. This enzyme is isolated from the thermophilic bacterium, Termus
aquaticus, and can survive extended incubation at 95 C. The Taq polymerase lacks editing
functions, and incorporates an incorrect base at a rate of 210-4 nucleotides pr. cycle in
polymerase chain reactions. The error frequency appears to increase in the presence of higher
concentrations of Mg2+ and dNTPs.
Reagents
10xPCR buffer
500 mM KCl
100 mM Tris-HCl pH 8.4
15 mM MgCl2
0.01 % gelatin
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Other reagents
dNTP
upper primer
lower primer
DNA Taq polymerase
3 Methods
Protocol
1. Purify the DNA fragment with agarose gel electrophoresis (page 40) followed by PCR
Preps DNA Purification System (page 41) prior to the PCR reaction.
2. Dilute the upper and the lower primers to 15 M final concentration with dH20.
3. Mix the PCR reaction mixture shown beneath in a small eppendorf tube, and aliquot it
into 5 eppendorf tubes.
25 l
(5 l)
5 l
(1 l)
8,5 l (1,7 l)
8,5 l (1,7 l)
5 l
(1 l)
173 l (34,6 l)
10 x PCR-buffer
10 mM dNTP
15 M upper primer
15 M lower primer
Taq DNA-polymerase
ddH2O until 45 l ( 225 l )
(each PCR tube)
4. Add 1, 2, 3, 4 or 5 l purified DNA fragment in each of the tubes containing the PCR
reaction mixture.
5. Put the tubes in the PCR apparatus, and start the reaction with the settings beneath
(programme 54).
3 min 94 C
1 min 94 C
1 min 58 C
1 min 72 C
x 40 cycles
4 C
6. Analyse an aliquot of the PCR products by agarose gel electrophoresis (page 40).
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3.2 DNA cloning in E. coli
3.2.1 Growth of E. coli
(Sambrook et al., 1989, page A.1)
E. coli is a colon bacteria with minimal growth requirements. Optimal growth temperature is 37
C, which is the temperature in the colon. The generation time is dependent on the bacteria strain
and growth phase, and varies from 20 minutes to hours.
LB medium
LB agar
1 % (w/v) Bacto-tryptone
0,5 % (w/v) Bacto-yeast extract
1 % (w/v) NaCl
dH2O until 1000 ml
Adjust pH to 7.0 with NaOH
Sterilise by autoclaving.
LB-medium with 1.5 % (w/v) agar
Sterilise by autoclaving
3.2.2 Frozen stock of E. coli
Protocol
1. Inoculate a bacterial colony in LB medium, or use an aliquot of culture made for maxiprep isolation (see page 48).
2. Incubate with shaking at 37 C until next day.
3. Mix 850 l bacteria culture with 150 l 85 % glycerol in an eppendorf tube.
4. Freeze rapidly. Store at – 70 C.
3.2.3 Transformation of E. coli
(Sambrook et al., 1989, page 1.74-1.85)
(Original TA Cloning Kit, Invitrogen)
Transformation is a process where bacteria (E. coli) takes up free DNA from solution. Linear
DNA fragments must be incorporated in the bacteria genome for replication. Plasmids, on the
other hand, replicate by themselves, by acting as extra circular genomes.
Transformed cells have to be selected from other cells in the transformation mixture. To ensure
this, plasmids contain markers, usually a gene encoding resistance to an antibiotic. Transformed
cells are selected by growing the transformation mixture in medium containing the same
antibiotic, where only transformed cells are able to live and multiply. During ligation, religation
of the plasmid can be a problem. To minimise the possibility for picking cells transformed with
an incorrectly inserted vector, the transformed cells are selected by the used of the complementation system.
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-Complementation
Selection of bacteria producing recombinant plasmid is done by -complementation. The vector
carries a short DNA segment containing the regulatory sequence and the coding information of
the first 146 amino acid sequence in the -galactosidase gene. Within this region is a polycloning
site that does not interfere with the enzyme activity of the produced N-terminal seguence of the
-galactosidase enzyme. The genome in the E. coli strain codes for the carboxyl-terminal
sequence of the -galactosidase gene. Neither of these fragments are themselves active, but if
they associate they form an active enzyme. This type of complementation is called complementation.
Insertion of a fragment in the polycloning site of the plasmid interferes with the production of the
N-terminal fragment making it unable of -complementation. An active enzyme hydrolyses the
hydroxylic bond in 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal), making a blue
product. As a consequence, a white colony on an agar plate represents bacteria containing
plasmid with insert, and a blue colony represents bacteria with religated plasmids without insert.
Reagents
SOC medium
20 g
Bacto-tryptone
5 g Bacto-yeast extract
0.5 g NaCl
0.19 g KCl
0.95 g MgCl2
20 mM glucose
dH2O until 1 L.
Sterilise by autoclaving
(Sambrook et al., 1989, page A.2)
Protocol
1. Mix gently 2 l 0,5 M -mercaptoethanol and 50 l One Shot INVF competent cells and
place the vial on ice. Add 2 l ligation reaction (page 39), and place on ice for 30
minutes. Treat the competent cells with care!
The plasmids adhere to the cell wall during incubation
2. Heat shock for 30 seconds at 42 C, and then place on ice for 2 minutes.
The plasmids enter bacteria through pores in the bacterial cell wall during the heat shock.
3. Add 450 l SOC medium (at room temperature).
4. Shake the tube horizontally at 37 C for 1 hour.
The bacteria repair the cell wall.
5. Spread 50 and 200 l from each transformation tube on separate LB agar plates with
antibiotic (50 g/ml ampicillin) and 50 g/ml X-gal. Make sure that the liquid is
absorbed, and incubate in incubator at 37 C until next day.
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3 Methods
Check for positively transformed bacteria (white colonies)
6. Inoculate a bacterial colony in 1 ml LB medium containing antibiotic (50 g/ml
ampicillin), and incubate at 37 C until next day.
7. Isolate plasmid DNA by mini prep isolation (page 46).
8. Treat with restriction enzymes (page 38) and separate by agarose gel electrophoresis
(page 40). Determine the insert size by comparison against a molecular standard to verify
successful ligation.
3.2.4 Mini preparation of plamid DNA
(Sambrook et al., 1989, page 1.25-1.31)
This method is for preparation of small plasmid DNA by alkaline lysis. The bacteria are
suspended in an iso-osmotic solution, and then treated with EDTA to break down the bacterial
cell wall (Solution I). The detergent SDS, in alkaline solution (Solution 2), lyses the resulting
spheroplasts. The alkaline denatures the DNA, and separation of bacterial DNA from plasmid
DNA is based on their ability to renature after the denaturation. Closed circular plasmid DNA
strands are unable to separate from each other because they are topologically intertwined. When
the pH is returned to normal, the strands of the plasmid DNA rapidly renature and form
superhelical DNA. Bacterial chromosomal DNA does not renature and is centrifuged out of
solution together with proteins and other cell components.
Reagents
Solution I
50 mM glucose
25 mM Tris-HCl (pH 8.0)
10 mM EDTA (pH 8.0)
Solution II
0,2 M NaOH
1 % (w/v) SDS
Solution III
5 M potassium acetate
11,5 % (w/v) acetic acid
H2O
60 ml
11.5 ml
28.5 ml
TE buffer
10 mM Tris-HCl (pH 8.0)
1 mM EDTA (pH 8.0)
Protocol
1. Transfer one single bacterial colony into 2 ml LB-medium containing antibiotic (50 g/ml
ampicillin), and incubate overnight at 37 C with vigorous shaking.
Harvesting
2. Transfer 1.5 ml of the bacterial culture into an eppendorf tube and centrifuge for 30
seconds at 4C in a microfuge. Store the remainder of the culture at 4 C.
3. Remove the supernatant and leave the pellet as dry as possible.
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Lysis by alkali
4. Resuspend the bacterial pellet in 100 l of ice
cold Solution I by vigorous mixing. It is essential
that the bacterial pellet is completely dispersed in
Solution I. This can be achieved rapidly by
vortexing two tubes simultaneously with their
bases touching each other (Figure 3-3).
Figure 3-3: Mixing of bacterial pellet.
5. Add 200 l freshly prepared Solution II to the resuspended bacterial pellet. Close the
tube tightly and mix by inverting the tube a few times. It is important that the entire
surface of the tube comes in contact with Solution II. Store the tube on ice.
6. Add 150 l ice cold Solution III and shake the tube gently for ten seconds to disperse
Solution III through the bacterial lysate. Store the tube on ice for 3-5 minutes.
Purification of the plasmid DNA
7. Centrifuge the tube at 12.000xg for 5 minutes at 4 C in a microfuge. Transfer the
supernatant to a fresh tube.
8. Add 500 l phenol:chloroform (250 l of each), and mix the solution by vortexing. After
centrifugation, transfer the supernatant to a fresh tube. Repeat extraction once, with
chloroform alone.
9. Add 2 x volume (800 l) ice-cold ethanol, and mix the solution by vortexing. Precipitate
the double stranded DNA at –20 C for 10 minutes.
10. Immediately after the precipitation, centrifuge the tube at 12.000xg at 4C for 5 minutes
in a microfuge.
11. Remove as much of the supernatant as possible. (Place the tube in an inverted position on
a sterile paper towel to allow all the fluid to drain away, and use a 40-200 l pipette to
remove any drops of fluid adhering to the walls of the tube).
12. Add 1 ml 70 % ethanol at 4 C to the pellet, and remove the supernatant as described in
step 11. Allow the pellet to dry in air for 10 minutes.
13. Redissolve the nucleic acid in 50 l TE-buffer containing 20 g/ml DNAase-free
pancreatic RNAase. Vortex briefly, and store at – 20 C.
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3.2.5 Maxi preparation of plamid DNA
(Qiagen Plasmid Handbook 02/95)
Maxi preparation of DNA is used for isolation of highly purified large amounts of DNA. The
isolation procedure is in principle similar to the mini preparation of DNA (page 46), but the
purification steps are different. Instead of phenol:chloroform extraction, the DNA is purified on
an anion exchanger in the first purification step. The DNA is then precipitated with isopropanol,
washed, dried and dissolved in TE-buffer.
Reagents
All solutions are included in the Qiagen Plasmid Maxi Preparation kit.
Protocol
Harvesting
1. Transfer one single plasmid containing bacterial colony into 500 ml LB-medium
containing 50 g/ml ampicillin. Grow bacteria overnight at 37 C with vigorous shaking.
2. Split the bacterial medium in 150-200 ml centrifugation tubes. Do not use more than 150
ml medium pr. Tube. Centrifuge at 20.000g for 30 minutes at 4 C, and remove the
supernatant.
Lysis of bacterial cells
3. Resuspend the bacterial pellet in 10 ml cold buffer P1.
4. Add 10 ml of buffer P2, mix the solution gently, and incubate at room temperature for 5
minutes.
5. Add 10 ml cold buffer P3, mix the solution immediately, but gently, and incubate on ice
for 15-20 minutes.
Purification of plasmid DNA
6. Centrifuge the solutions at 20.000g for 30 minutes at 4 C. Transfer the supernatant
immediately to another tube, and centrifuge again at 20.000g, but for 15 minutes at 4 C.
Store the supernatant on ice.
7. Equilibrate a Qiagen-tip 500 for each centrifugation tube by applying 10 ml buffer QBT.
Allow the column to empty by gravity force.
8. Apply the supernatant from step 6 to the Qiagen-tip and allow it to enter the resin by
gravity flow.
9. Wash the Qiagen-tip twice with 10 ml buffer QC, and elute with 15 ml buffer QF.
10. Precipitate the DNA with 0.7 volumes of isopropanol at room temperature, and centrifuge
immediately at 15.000g for 30 minutes at 4 C. Remove the supernatant carefully.
11. Wash the DNA pellet with 70 % ethanol, air-dry for 5 minutes, and redissolve in 300 l
TE-buffer.
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3.3 mRNA Analysis
Detection of specific mRNA is often used to verify the expression of an active gene in a cell,
since transcription of DNA to mRNA in a cell is the first product of the active gene. Northern
blot is often used for identification of the mRNA. The northern blot analysis is a reliable and thus
widely used method, even if it involves several steps (Figure 3-4).
Isolation of mRNA with Dynabeads Oligo (dT)25
Separation of mRNA by agarose gel electrophoresis
Hybridization with radioactive probe
Northern
Transfer of mRNA to nylon membrane
Detection of specific mRNA
Figure 3-4: Northern blot analysis
A flow chart presenting the different steps in a Northern blot analysis.
RNA is an unstable molecule, highly vulnerable for degradation by RNase activity coming from
the hands of the worker, the lysed cells, working solutions or the equipment used in the isolation
process. Inhibiting or avoiding RNases is absolutely necessary when isolating RNA.



Equipment must be treated with 7 % acetic acid, RNase AWAY or heated at 180C
until next day.
Distilled water is incubated with 0.1 % (w/v) diethyl-pyro-carbonate (DEPC) until
next day, and autoclaved at 120C for 20 minutes.
Solutions are made of RNase free chemicals diluted in DEPC-treated water.
Protocols describing each step during the Northern blot analysis are not included - only the
principles in each method is presented. A complete description of the methods can be found in
another thesis (Mustorp, S. L., 1997).
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3.3.1 Purification of mRNA with Dynabeads Oligo (dT)25
(Biomagnetic Techniques in Molecular Biology, Dynal)
All eucaryotic cells have a polyA sequence in the 3`end of their mRNAs. Dynabeads Oligo
(dT)25 is designed for rapid isolation of highly purified intact polyA mRNA by binding to this
tail. The magnetic Dynabeads contain covalently attached Oligo (dT)25, which hybridises to the
polyA tail on the mRNA. During the washing steps, the complex is separated from the remaining
solution by a magnet, while DNA, proteins and other macromolecules are washed away (Figure
3-5). In the end, the mRNA is eluted by heating the solution, which separates the mRNA and the
Oligo (dT)25.
DNA
Cells are lysed
by sonication
mRNA
AAAAAAAAA
A
Protein
TTTTTTTTT
Dynabeads Oligo (dT)25
Dynabeads
Oligo
(dT)25 is added to the
cell suspension.
The mRNA binds to the
Dynabeads Oligo (dT)25.
TTTTTTTTT
AAAAAAAA
A
The mRNA-Dynabeads Oligo (dT)25complex is held back by a magnet during
the washing steps.
Protein
DNA
TTTTTTTTT
AAAAAAAA
A
The mRNA is separated from the
oligo (dT) beads in elution buffer.
mRNA
AAAAAAAA
A
TTTTTTTTT
Figure 3-5: mRNA isolation with Dynabeads Oligo(dT)25 magnetic beads.
Cells are lysed and the suspension is mixed with Dynabeads Oligo(dT)25. The poly-T arm binds to poly-A tail in
mRNA with hydrogenic bonds. During the washing steps, the complex is retained on the eppendorf tube wall by a
magnet outside the tube. After the washing steps, the mRNA is eluted from the Oligo(dT) 25 by breaking the
hydrogenic bonds with heating.
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3.3.2 Northern Analysis
Agarose electrophoresis of mRNA
(Sambrook et al., 1989, page 7.43)
As described for electrophoresis of DNA (page 40), negatively charged phosphate groups in
mRNA make it electric mobile, migrating towards the anode in an electric field. The separation
of mRNA is carried out in an agarose gel containing formaldehyde to denaturate secondary
structures in mRNA. When denatured, the mRNA is separated due to molecular size only. For
optimal separation, the electrophoresis is carried out at 4C, with high voltage - 5.6 V/cm (140 V
in the RNA electrophoresis chamber). Due to the high voltage, the agarose gel is heated during
electrophoresis, and may melt at room temperature.
Diffusion blot
(Sambrook et al., 1989, page 7.46)
The mRNA is transferred immediately after electrophoresis to a nylon membrane (Hybond N)
with capillary diffusion (Figure 3-6). The gel containing formaldehyde must be rinsed several
times in DEPC-treated water and blotting buffer (20 x SSC) to remove formaldehyde. After
blotting, the mRNA is fixed to the membrane by baking the membrane at 80C for 2 hours.
Weight
Glass plate
Paper towels
Whatman 3 MM paper
Nylon membrane
Gel
Support
Whatman 3 MM paper
Figure 3-6: Capillary diffusion of mRNA from an agarose gel to a nylon membrane.
Radioactive labelling of cDNA
(Sambrook et al., 1989, page 10.14)
The radioactive probe, used to detect specific mRNA is made by mixing denaturated (by boiling)
purified cDNA, with heterogeneous hexo-deoxynucleotide primers. Synthesise of [32P]-labelled
probes is carried out by using the Klenow fragment of E. coli DNA polymerase I. One of the
nucleotides used for incorporation is labelled ([32P]dNTP) while the others are unlabeled.
Unincorporated nucleotides are removed by a spin column (Probe Quant G-50 Micro Column)
before hybridisation.
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3 Methods
Isolation of cDNA
Isolate plasmids (containing the cDNA) from E.coli using a plasmid isolation procedure (page 46
or 48), cut out the cDNA fragment with restriction enzymes (page 38), purify the fragment by
agarose electrophoresis (page 40) and remove the agarose with WizardTM PCR preps DNA
purification Systems (page 42).
Church hybridisation
(Church and Gilbert, 1984)
In the hybridisation process, the labelled probe is used to detect the mRNA of interest on the
membrane. The nylon membrane is incubated together with the 32P-labelled denatured cDNA
probe. Hydrogen bounds are formed between the cDNA and mRNA on the membrane
(hybridisation). At optimal temperature, the probe binds specifically and stronger to the mRNA
with complementary sequence than to random mRNA. Non-specifically bound cDNA is removed
by washing the membrane after hybridisation.
Detection of specific mRNA is done in PhosphorImager. Cover the membrane with plastic and
expose the screen plate for 2-24 hours.
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3.4 Cell lines in culture
3.4.1 MDCK II cell line
The Madin-Darby canine kidney cell line was isolated from a male canine kidney by Madin and
Darby in 1958, and used frequently for several years before it was characterised (Gaush et al.,
1966). Two sublines of MDCK, both distinct and stable in culture, have been characterised
(Barker and Simmons, 1981; Richardson et al., 1981). The MDCK I cell line, a subclone of
MDCK II, resembles tight epithelia, whereas the MDCK II cell line forms more leaky epithelia
(Barker and Simmons, 1981; Richardson et al., 1981). In this thesis, the MDCK II cell line is
used. The Madin-Darby canine kidney cell line (MDCK II) is though to derive from the proximal
convoluted tubule of the kidney.
Reagents
Trypsin solution
8,0 g
0,2 g
0,2 g
0,2 g
1,2 g
2,5 g
NaCl
KH2PO4
KCl
EDTA
Na2HPO4
Trypsin
Add redistillated water to 1000 ml.
(the solution should have pH 7.4).
Incubate the solution for 1 hour at 37 C. Do not shake.
The trypsin is dissolved when the solution is almost clear.
Sterile filtrate the solution and store at – 20 C.
Complete medium
MDCK II cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with
5 % fetal calf serum (FCS), 100 IU/ml penicillin, 50 g/ml streptomycin and 2 mM L-glutamine.
Splitting of cells (75 cm2 flasks)
Split the MDCK II cells 1:6 at confluent, every 3-4 days. The different serglycin producing
MDCK II clones need longer incubation time with trypsin than wild type MDCK II cells. For
unknown reasons, there seems to be a correlation between the synthesis and secretion of the
recombinant serglycin-His-Flag, and the resistance to the trypsin solution.
1. Preincubate complete medium and trypsin solution at 37 C.
2. Remove the medium from the flask with sucktion coupled to a vacuum pump.
3. Wash the cells with 8-10 ml trypsin solution for 1-2 minutes.
4. Remowe the trypsin solution.
5. Add 2 ml trypsin solution, and incubate the flask at 37 C until the cells have come off
from the plastic.
6. Resuspend in 10 ml complete medium, and mix gently. Wash the whole bottom of the
flask when mixing.
7. Pipette 2 ml from this solution to a new 75 cm2 flask with 20 ml complete medium.
8. Place in an incubator at 37C with 5 % CO2.
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3 Methods
3.4.2 Polarised culturing and labelling of MDCK II cells
In vivo, epithelial cells are polarised, with two different functional domains. It is thus wanted to
recreate this polarisation, when studying various cellular processes in the epithelial cells. By
culturing the MDCK II cells on filter membranes, the cells resemble the in vivo situation and
form a confluent monolayer composed of morphological and functional polarised cells with
apical and basolateral domains (Barker and Simmons, 1981).
The cells are seeded onto filter membranes, which contain pores that are large enough to let
molecules pass through, but too small to let the cells migrate through. After seeding, the cells
grow on top of the filter, and form tight junctions near the apical surface of the cell layer. The
tight junctions connect the cells, and the confluent cell layer forms a tight barrier that separates
the medium into two compartments, the apical and the basolateral. Due to this layer, molecules
are not allowed to pass from one compartment to another without active transport through the
cell layer (Figure 3-7). The MDCK II cell line has thus been used frequently when studying
cellular processes in epithelial cells, and is so far the best characterised epithelial cell line
(Simons and Fuller, 1985).
Apical medium
Confluent cells
Tight junction
Apical
Lateral
Filter
memb
rane
Well
Basal
Basolateral medium
Figure 3-7: Polarised culture of MDCK II cells on filter membranes.
The glycosaminoglycan (GAG) chains in the proteoglycans are highly substituted with sulphate
during post-translational modifications in the Golgi apparatus. By substitution of cold sulphate in
the medium with 35(S)sulphate, the proteoglycans can be radioactively labelled to increase the
detection limit in later experiments. Low substitution of 35(S)sulphate into other molecules
produced by the cell, provides (35S)sulphate labelling more or less specific for proteoglycans.
Free (35S)sulphate added to the medium is transported through the cell membrane and is
activated in the cytosol by the formation of adenosine 3'-phosphate 5'-phosphosulphate (PAPS).
PAPS is transferred into the lumen of the Golgi apparatus by the PAPS transporter, and then used
as substrate by various sulphotransferases in the Golgi lumen.
54
3 Methods
Procedure
Polarised culturing on filter membranes
1. Put a 24 mm Transwell polycarbonate filter membrane into a filter adapter, and place it at
the bottom of a 15 cm petri dish (see beneath). (Do not use more than 6 filters in the same
petri dish). Add 90 ml complete medium (basolateral medium).
Filter membrane
Filter adapter
Petri dish
2. Split a confluent 75 cm2 flask as described on page 53. Transfer the cell suspension to a
50 ml Falcon tube and centrifuge at 1000 rpm for 5 minutes.
3. Remove the supernatant and resuspend the cells in 10 ml complete medium.
4. Pipette 1,6 ml suspended cells on top of each filter membrane (apical medium).
5. Incubate the cells for 4 days, to make the cell layer confluent. The cells are ready for
experiments.
Labelling with (35S)sulphate
6. For each membrane, fill a 24 mm well with 2 ml labelling medium (0.2 mCi/ml
(35S)sulphate in sulphate free RPMI medium with 5 % fetal calf serum, 100 IU/ml
penicillin, 50 g/ml streptomycin and 2 mM L-glutamine).
7. Loosen the filter membrane containing the confluent cell layer from the filter adapter, and
remove the apical medium.
8. Add 1 ml labelling medium on top of the cell layer (apical medium), and then put the ring
into the well containing 2 ml labelling medium (basolateral medium).
9. After 20 hours incubation, harvest apical and basolateral medium separately. Centrifuge
the apical medium at 1000 rpm for 5 minutes to remove loosen cells, and transfer the
supernatant to a new tube.
10. Remove unincorporated sulphate with either DEAE anion exchange chromatography
(method 3.7.2, page 69), or Sephadex G-50 Fine chromatography (method 3.7.3, page
70).
55
3 Methods
3.4.3 HAEC cell line
(Product sheet, Cascade Biologics, INC.)
The human aortic endothelial cell line (HAEC) is derived from normal aorta. The cells express
human factor VIII antigen, but not -antigen. For proper growth, the HAEC cells require low
serum growth supplement (LSGS), which contains all of the growth factors, hormones and serum
necessary for the growth of human endothelial cells, derived from large vessels. Addition of
serum is thus not necessary.
Reagents
Trypsin/EDTA solution (R-001)
Trypsin neutraliser (R-002)
Complete medium
Human aortic endothelial cells (HAEC) were cultured in Medium 200 (M200) with Low Serum
Growth Supplement (LSGS), 100 IU/ml penicillin and 50 g/ml streptomycin.
Splitting of cells (75 cm2 flasks)
1. Withdraw the medium from the flask with a vacuum pump.
2. Add 10 ml Trypsin/EDTA solution to the flask. Incubate at room temperature for 2
minutes.
3. When the cells have become completely round, slap the flask gently to loosen the cells
from the surface of the flask.
4. Add 5 ml Trypsin Neutraliser, mix gently and transfer the suspension to a 15 ml tube.
5. Centrifuge the cells at 1000 rpm for 5 minutes.
6. Remove the supernatant form the tube and resuspend the cell pellet in 10 ml M200.
7. Transfer 2 ml cell suspension to a new 75 cm2 flask and dilute with complete medium
until 15 ml medium.
8. Incubate the HAEC cell line at 37 C with 5 % CO2.
56
3 Methods
3.4.4 HUVEC cell line
(Product sheet, ATCC)
The human umbilical vein endothelial cell line (HUVEC), is derived from the vein of a normal
human umbilical cord. The cells express human factor VIII antigen, and have a life expectancy of
50 - 60 populations. Endothelial cell growth supplement (ECGS), and unidentified factors from
bovine pituitary, hypothalamus or whole brain extracts are mitogenic for this cell line.
Reagents
Trypsin solution
Use the same trypsin solution as for MDCK II cells.
Complete medium
Human umbilical vein endothelial cells (HUVEC) were cultured in Endothelial-SFM Medium
with Endothelial Cell Growth Supplement (ECGS), 10 % fetal calf serum (FCS), 100 IU/ml
penicillin and 50 g/ml streptomycin.
Splitting of cells (75 cm2 flasks)
Coating of flask
1. Incubate gelatin at room temperature or 37 C, if the gelatin solution is viscous.
2. Add gelatin solution and wet the whole bottom of the flask. Transfer the gelatin solution
back to the bottle for reuse.
3. Enable the gelatin to dry before adding the cell suspension.
Transfer cells to new flask
4. Incubate complete medium and trypsin solution at 37 C.
5. Remove the medium from the flask with a vacuum pump.
6. Wash the cells with 3 ml trypsin solution.
7. Remove the washing solution.
8. Add 1.5 ml trypsin solution, and incubate the flask at 37 C until the cells have come off
from the plastic. This should take ½ to 1 minute.
9. Add 5 ml complete medium, mix gently and wash the whole bottom of the flask when
mixing. Transfer the cell suspension to a 15 ml Falcon tube.
10. Centrifuge the cells at 1000 rpm for 5 minutes.
11. Remove the supernatant.
12. Resuspend the cell pellet in 5 ml complete medium and count the cell number. Dilute the
cells to desired density (1:2) and transfer to the coated flask. Add medium until 15 ml in
total.
13. Incubate the HUVEC cell line at 37 C with 5 % CO2.
57
3 Methods
3.4.5 U937-1 cell line
The human monoblast cell line U937-1 is a subclone of the U937 cell line isolated from a patient
with histiocytic lymphoma. It shows characters common to cells on the monoblastic stage
(Sundstrøm and Nilson, 1976). The U937-1 cell line produces only chondroitin sulphate
proteoglycans (Kolset et al., 1996), and N-terminal sequencing has shown that the secreted
proteoglycan produced by U937 is serglycin (Uhlin-Hansen et al., 1993). The U937 cell line was
therefore used as control during mRNA analysis, and as a PG source in some experiments.
Reagents
Complete medium
U937-1 cells were cultured in RPMI 1640 medium with 10 % fetal calf serum (FCS), 100 IU/ml
penicillin, 50 g/ml streptomycin and 2 mM L-glutamine.
Splitting of cells (75 cm2 flasks)
1. Transfer the cell suspension to a 50 ml tube. Centrifuge at 1000 rpm for 5 minutes.
2. Remove the supernatant, resuspend the cells in 10 ml medium and count the cell number.
3. Seed the cells with a density of 0,3  106 cells/ml in 20 ml medium.
4. Incubate the U937 cell line at 37 C with 5 % CO2.
3.4.6 Labelling with (35S)sulphate (HAEC, HUVEC and U937 cells)
Procedure
1. Culture cells as described for the cell line.
2. Remove the medium and wash once with DPBS.
3. Incubate cells with 0.1-0.2 mCi/ml (35S)sulphate in complete medium for 20 hours. Use
sulphate free medium if possible, which increases the labelling efficiency about ten-fold.
4. Remove unincorporated 35(S)sulphate with either Sephadex G-50 Fine chromatography
(page 70) or DEAE anion exchange chromatography (page 69).
58
3 Methods
3.5 Transfection
(Ausubel et al., 1996, unit 9.1)
Transfection is a method for introducing foreign DNA into mammalian cells. The four most
common methods are calcium phosphate transfection, DEAE-dextran transfection,
electroporation and liposome mediated transfection. Cells show variation in transfection
efficiency with different methods, making it difficult to find the best-suited method for
transfection of a particular cell line. The calcium phosphate transfection method was used to
transfect MDCK II cells, and both calcium phosphate and liposome mediated transfection
methods were tried out for transfection of endothelial cells.
The best way to optimise a transfection method is to transfect the cells with an easily detectable
reporter gene. A plasmid containing green fluorescent protein (GFP) is well suited for such
studies. GFP will give fluorescence of green light (507 nm) when excitated by blue light (488
nm). When viewed in a fluorescence microscope, transfected cells that produce satisfactory
amounts of GFP will give fluorescent green light, strong enough to be detected by eye.
There are two main types of transfection, transient transfection and stable transfection. In
transient transfection studies, the gene product or some biochemical reaction(s) is analysed when
the cDNA has entered the nucleus and is expressed, usually some days after transfection. High
transfection efficiency is then important to see the biological effect of the gene. During
transfection, approximately one of 104 cells stably integrate the foreign DNA into the genome.
Stably transfected cells can be isolated from other cells with a positive selectable marker and
used for further studies. For our experiments, it was necessary to obtain stably transfected cells.
3.5.1 Tolerance test for geneticin (G-418)
(Ausubel et al., 1996, unit 9.4, Supplement 36)
The pcDNA3.1(-)/Myc-His vector contains the bacterial aminoglycoside phosphotransferase
gene (APH), which detoxificate geneticin (G-418). Cells stably transfected with the vector
express APH and are able to divide in medium containing geneticin (Figure 3-8). Geneticin
blocks protein synthesis in mammalian cells by interfering with ribosomal function. It is an
aminoglycoside, similar in structure to neomycin, gentamycin and kanamycin. Cells differ in
their susceptibility to geneticin, and the concentration in the selection medium must be
determined for each cell line prior to transfection.
CH3
HO
C
H
O
H
H
OH
H
H
NH2
HO
H
NH2
H
H
OH
H
H
H
NH2
O
H
H
O
H
O
HO
NHCH3
H
H
H3C
H
H
Figure 3-8: The structure of geneticin (G-418)
Geneticin should be diluted in a strong buffer, (e.g., HEPES, pH 7,3), so
that addition of the drug does not alter the pH of the medium.
OH
59
3 Methods
Procedure
1. Seed one 12 well plate with wild type cells in 1 ml standard medium, and grow cells to
70-80 % confluency.
2. Make selection medium by adding 600, 300, 150, 100, 50 or 10 g/ml G-418 to complete
medium. (Use two parallels for each concentration).
3. Replace the selection medium every 3 days for 9 days, and look at the cells in a
microscope.
Follow the cells in a microscope. Depending on the duration of the cell cycle for the cells, the
cells will start to die after five to seven days in wells with too high G-418 concentration. Five
days later, cells in wells with lower concentration will also die. Wells with living cells have been
given too low concentrations. Define the selection medium for each cell line by doubling the
lowest concentration of G-418 killing all cells after nine days.
Selection medium for MDCK II cells: Geneticin concentration of 500 g/ml
(Norgeng, T, personal communication).
Selection medium for HUVEC and HEAC cells: Geneticin concentration of 100 g/ml.
3.5.2 Transfection of MDCK II cells (calcium phosphate method)
(Ausubel et al., 1996, unit 9.1, Supplement 36)
(Nordeng, T, personal communication, published in: Simonsen et al., 1997)
In the calcium phosphate method, the DNA is introduced to the cells via a precipitate that
adheres to the cell surface. A HEPES buffer is used to form a calcium phosphate and DNA
precipitate that is directly layered onto the cells. Depending on the cell type, up to 10 % of the
cells take up the DNA precipitate through an undetermined mechanism. Shocking the cells with
dimethyl sulphoxide (DMSO) can improve the transfection efficiency. Some cells are stressed by
the calcium phosphate method, and do not recover, due to the high DNA-calcium phosphate
precipitate concentration needed for efficiently introduction of the DNA.
The calcium phosphate method may be used for both transient and stable transfection of adherent
cells. It introduces large amounts of DNA into the cells that pick up the DNA, which increase the
possibility that some of it will be stably transfected.
The MDCK II cell line was successfully transfected with the calcium phosphate method. The
HUVEC and the HAEC cell lines were stressed and did not recover after transfection with this
method. Lower precipitation concentrations and incubation time were tried, which only resulted
in very low transfection efficiencies.
Reagents
2 x HEPES buffer
0.28 M NaCl
(16,4 g)
0.05 M HEPES
(11,9 g)
1.5 mM Na2HPO4 (0.21 g)
800 ml dH2O
Titrate to pH 7.05 with 5 M NaOH
Add H2O to 1 liter.
Filter sterilise (0.45 m)
60
2.5 M CaCl2
183,7 g CaCl2 2H2O
dH2O to 500 ml
Filter sterilise (0.45 m)
3 Methods
Procedure
Seeding
1. Seed 3  105 cells (1:20 confluent 75 cm2 flask) on a 10 cm petri dish one day prior to
transfection in 10 ml complete medium.
Transfection
2. Suspend 20 g transfection vector (pcDNA3.1(serglycin)/Myc-His vector 1, 4 and 7) for
each transfection in 100 l dH2O in a 1.5 ml eppendorf tube.
3. Mix 350 l dH2O and 50 l 2.5 M CaCl2 in another eppendorf tube, and transfer gently
the diluted vector to this tube (0.25 M CaCl2, final concentration).
4. Pipette 500 l 2 x HEPES in another 1.5 ml eppendorf tube.
5. Add the DNA/CaCl2-solution to the HEPES-buffer. Use one mechanical pipettor to
bubble the HEPES-buffer, and another one to add the DNA/CaCl2-solution drop wise
(Figure 3-9). It is important not to add the DNA/CaCl2-solution to rapidly to prevent
formation of large precipitate, which will lower transfection efficiency. Incubate the
solution for 10-15 minutes at room temperature.
Pipettor adding the
DNA/CaCl2-solution
Pipettor making
air bubbles
HEPES buffer
Figure 3-9: Mixing of DNA/CaCl2 solution with HEPES buffer.
For high transfection efficiency, it is important not to add the DNA/CaCl 2 solution to
rapidly. One pipettor is used to bubble the HEPES buffer while the other is used to
add the DNA/CaCl2 solution dropwise.
6. Distribute the DNA precipitate over the entire area of the cells, and incubate the petri
dish at 37 C with 5 % CO2 until next day (ca. 20 hours).
7. Remove the medium the morning after, and wash the cells with 10 ml PBS.
DMSO shock
8. Add 1 ml cold 10 % DMSO in PBS at the edge of the pertri dish and distribute gently
over the cells. Let sit for 5 minutes at room temperature.
61
3 Methods
9. Add 10 ml PBS onto the cells. Remove the fluid and add 10 ml complete medium. Place
the dish in CO2 incubator for 1 hour.
10. Replace with fresh complete medium, and let the cells grown for 24 hour before using
selection medium.
Splitting of cells for selection of stabile transfected cells.
11. Split cells 1:100, 1:30, 1:10 and 9:10 (rest) in four petri dishes. Add 10 ml selection
medium with proper concentration (500 g/ml G-418 for MDCK II cells). As a control,
give non-transfected cells selection medium with the same G-418 concentration.
12. Replace with selection medium every two to three days for 9 days, then every five days.
13. When all control cells are dead, select stably transfected cells (method 3.5.4, page 63).
3.5.3 Transfection of HAEC cells (lipofectin method)
(Ausubel et al., 1996, unit 9.4, Supplement 36)
(Lipofectin transfection protocol)
The lipofectin reagent is a mixture of polycationic liposomes. Use of liposomes to deliver DNA
into eucaryotic cells results in higher efficiency and greater reproducibility than other transfection
methods. Relatively small amounts of DNA are effectively taken up and expressed. Liposomes
are therefore the best choice for transient expression.
In the lipofectin method, plasmid DNA is mixed with the liposome mixture and applied to a
monolayer of adherent cells. Three primary parameters affect the success of the transfection - the
concentration of DNA, the concentration of lipofectin and the incubation time. Generally,
increase of all parameters, up to a level where they become toxic to the cells, improves the
transfection efficiency. Transfection efficiency is also better using cells with low passage
(preferably not beyond passage 6).
The HAEC cells were transfected with varying lipofectin and DNA concentrations and
incubation time. The protocol presented here represents the result obtained in the optimisation
study. Increase in DNA or lipofectin concentration or longer incubation times does not improve
transfection. With the settings beneath, one can expect transient transfection of 10 % of the
HAEC cells (fluorescent cells 2 days after transfection with pEGFP-N1 vector). Isolation of
stably transfected clones was not achieved.
The DPBS wash is not included in the original protocol, but this washing step prevented an
otherwise small cell death the day after transfection. DMSO or glycerol shock right after
transfection was not performed.
Reagents
Lipofectin reagent
OPTI-MEM medium (serum free medium)
Complete medium
62
3 Methods
Procedure
1. Seed 4  105 cells in 2 ml complete medium in a 24 mm well (6 well plate). This cell
density is obtained by splitting a 75 cm2 flask, and distribute the cells in two plates.
2. Incubate the cells to 60-70 % confluency at 37 C with 5 % CO2.
3. Prepare the following solutions in sterile 1,5 ml eppendorf tubes.
Solution A: For each transfection, dilute 5 g DNA in 100 l serum free medium
(OPTI-MEM I).
Solution B: For each transfection, dilute 8 to 10 l lipofectin (1g/l) in 100 l
serum free medium. (OPTI-MEM I).
4. Combine solution A and solution B, mix gently and incubate at room temperature for 15
minutes. (The solution could appear cloudy, but this will not affect the transfection).
5. Wash the cells two times with 2 ml DPBS, and add 0.8 ml serum free medium (OPTIMEM I) per cell well. Neither antibiotic agents nor serum should be used during
transfection.
6. Add the lipofectin/DNA solution on top of the cells, and mix gently.
7. Incubate the cells for two hours at 37 C with 5 % CO2.
8. Remove the transfection mixtures. Wash the cells two times with 2 ml DPBS, and add 2
ml complete medium pr. well (M-200 with supplement).
3.5.4 Isolation of stable transfected colonies using cloning rings
(Ausubel et al., 1996, unit 9.5, Supplement 36)
During transfection, approximately one of 104 cells stably integrate the foreign DNA into the
genome. A cDNA encoding a selective marker in the transfection vector, which gives resistance
to a toxic compound added to the medium, is used to permit isolation of the stable transfectants.
The cells should grow in selection medium for approximately 10 doublings before individual
colonies are picked and expanded into cell lines.
Procedure
1. Transfect the cells with one of the methods described previously (page 60 or 62), and
allow them to divide once before adding selection medium. Add the optimal
concentration of geneticin to the medium (page 59) and let the cells grow for 10 divisions
before colonies are picked. Replace the medium every third day.
2. Pick large single colonies after use of selection medium. (Small colonies may not contain
enough cells for proper growth).
3. Remove the medium and wash the cells with PBS.
4. Put a sterile cloning ring on top of a single colony and add PBS outside the ring(s) until
the bottom of the well is covered. Add 3 drops (150 l) of trypsin solution inside the ring
and incubate (30 minutes for MDCK II cells).
5. Mix the trypsin treated cells with a few drops of complete medium, and transfer the cells
to a small well (12 well plate). Allow the cells to grown to confluency, split the cells, and
then transfer the cells into larger wells/culture flasks.
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3 Methods
3.6 Analysis of glycosaminoglycans on proteoglycans
3.6.1 Chondroitinase ABC lyase treatment
(Modified method of Ausubel et al., 1994-1996, Chapter 17.13.23).
Chondroitin ABC lyase (cABC) from Proteus vulgaris, acts endolytically on chondroitin
sulphates A-E, slowly on hyaluronic acid, and not at all on heparin, heparan sulphate or keratan
sulphate. The enzyme cleaves the (14) glycosidic bond between N-Acetyl-D-galactosamine
sulphate and D-glucuronate or D-Iduronate. It cleaves any chondroitin sulphate or dermatan
sulphate chain, without a specific requirement for sulphating pattern for action, while other
chondroitin lyases require specific sulphation of the N-Acetyl-D-galactosamine for cleavage
(Figure 3-10.A). These are therefore better for structural determination of the saccharides in
chondroitin sulphate or dermatan sulphate chains. The cABC degrades chondroitin sulphate
chains into disaccharides, except for the linkage region, which remains attached to the core
protein after treatment (Figure 3-10.B).
Chondroitinase ABC
lyase treatment at
37C until next day.
A
B
Figure 3-10: Degradation of chondroitin sulphate by chondroitinase ABC lyase.
A: The cleavage site for chondroitin ABC lyase, and the sulphation pattern required for action. B: The reaction
products after chondroitinase ABC treatment of a chondroitin sulphate proteoglycan.
Reagents
10 x cABC buffer
50 mM Tris-HCl (pH 8.0)
50 mM Sodium acetate
0,1 % (w/v) BSA
Adjust pH to 8.0
64
3 Methods
Protocol
Mix cABC lyase (0,025 U/10.000 cpm) with sample and add 10x cABC buffer to final
concentration of 1x. Incubate at 37C until next day.
Comment:
The elution buffer after Ni2+-affinity chromatography (phosphate buffer with 100 mM imidazole) does not
inhibit the cABC lysase enzyme (tested on purified serglycin form U937 cells). Dialysis of sample or buffer
exchange before analysis is therefore not necessary.
3.6.2 Nitrous acid degradation (HNO2 treatment)
(Modified method of Ausubel et al., 1994-1996, 17.22).
There exists two protocols for HNO2 degradation of glycosaminoglycans. The first facilitates
cleavage of the glycosidic bond after N-sulphated glucosamine residues in a glycosaminoglycan
chain, and the other method, not present here, cleaves after a glucosamine residue in a
glycosaminoglycan chain. The main difference between these two protocols is the pH of the
HNO2 reagent used.
Heparin and heparan sulphate chain contains a mixture of both N-sulphated and N-acetylated
glucosamines, with N-sulphated glucosamine being most common. No other glycosaminoglycan
chain has N-sulphated glucosamines. Cleaving after N-sulphated glucosamines is therefore
specific for heparin and heparan sulphate (Figure 3-11).
When the polymer is treated with HNO2 at pH 1.5, it is cleaved without loss of O-sulphate
substituents to yield a mixture of oligosaccharides having 2,5 anhydro D-mannose residues at
their reducing terminals. Some polysaccharide units are formed due to the non-reactive Nacetylated glucosamines in the chain. Heparin is cleaved into smaller fragments, since it contains
more N-sulphated glucosamines than heparan sulphate.
Degradation of
heparan sulphate
Nitrous acid treatment
at pH 1.5
Degradation of
heparin
Figure 3-11: Nitrous acid degradation of proteoglycans with heparan sulphate glycosaminoglycan
chains.
Reagents
Nitrous acid reagent
0,5 M H2SO4
0,5 M Ba(NO2)2
Reaction neutraliser
1 M NaOH
(or 1 M Tris-HCl buffer, pH 8.0)
65
3 Methods
Protocol
1. Cool the H2SO4 and the Ba(NO2)2 solutions and all samples on ice.
2. Mix equal volumes of 0,5 M H2SO4 and 0,5 M Ba(NO2)2, and centrifuge the solution
until all BaSO4 has precipitated. Transfer the supernatant to a new eppendorf tube. This
reagent should be prepared when needed and used immediately.
3. Mix equal volumes of HNO2-reagent and sample, and incubate at room temperature for
10 minutes. Do not use too small volumes. At least 50 l of total reaction mixture is
recommended.
4. Neutralise the reaction mixture with 1 M NaOH until pH  7 (Measure the pH with pH
paper). Adjust pH if necessary.
3.6.3  -elimination of glycosaminoglycans (NaOH treatment)
(Modified method of Ausubel et al., 1994-1996, 17.15.1).
Most glycosaminoglycan (GAG) chains are O-linked to the core protein with an alkaline labile
xylosideserin linkage. These O-linked glycosaminoglycan (GAG) chains in proteoglycans are
readily released from their core proteins by treatment with alkali at room temperature. In the
reaction, the serine residues are converted to dehydroalanine residues, but the core protein is not
cleaved. Thus both the protein core and the released GAG chains are intact after nitrous acid
treatment (Figure 3-12). Their molecular weights can be estimated by gel filtration
chromatography (page 73) or SDS-PAGE (page 67).
NaOH treatment
Figure 3-12: -elimination of Glycosaminoglycan chains on proteoglycans.
N-linked oligosaccharides remain attached to the core protein, but any O-linked
oligosaccharides will be released together with the GAG chains.
Protocol
Perform the reaction in an eppendorf tube. Solve the sample in buffer with low salt
concentration.
1. Add 5 M NaOH until final concentration of 0,5 M NaOH. Incubate at room temperature
until next day.
2. Neutralise the solution and stop the reaction with 1 M HCl. (Use 5 times the volume of
NaOH.)
3. Measure the pH with pH-paper. The pH of the final solution should be in the range from 6
to 8. Adjust pH if necessary.
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3 Methods
3.7 Separation of proteoglycans
3.7.1 SDS-PAGE (Novex)
(Helgeland, L, KJ220, høst 96) and (NovexTM Precast Gel Instruction)
Polyacrylamide electrophoresis (PAGE)
Polyacrylamide gels is used to separate macromolecules. As in agarose gel electrophoresis (page
40), large molecules migrate slower in the matrix than small molecules. The gel matrix consists
of long polyacrylamide chains cross-linked with bis-acrylamide, where the pore size in the matrix
is determined by two factors:
The total concentration of acrylamide and bis-acrylamide (T)
Concentration of bis-acrylamide (C)
Regardless of the total concentration (T), the average pore size reaches a minimum when C is 5
%. The pore size is usually controlled by varying T while C is fixed at 5 %.
Polymerisation of the matrix is carried out by adding tetra-methylenediamine (TEMED) and
ammonium persulphate (APS) to the reaction mixture. When ammonium persulphate is
dissolved in water, it forms free radicals (S2O82-  2 SO4-). If a free radical is brought in contact
with acrylamide, the radical is transferred to the acrylamide molecule making it highly reactive.
This acrylamide then reacts with other acrylamides and produces a long polymer chain.
Formation of a matrix requires that these polymer chains are cross-linked to each other. This is
done by polymerisation in the presence of bis-acrylamide. TEMED acts as a catalyst of the gel
formation, due to its ability to exist in a free radical form.
Sodium dodecyl polyacrylamide electrophoresis (SDS-PAGE)
Proteins possesses charges as a result of acidic and basic amino acids, and in PAGE, the
migration depends on the protein charge. In sodium dodecyl sulphate-polyacrylamide
electrophoresis (SDS-PAGE) proteins are separated primarily by their molecular weights.
Negatively charged SDS molecules bind along the polypeptide chain and mask the charges in the
protein. During electrophoresis, the length of the reduced SDS-protein complex is proportional to
its molecular weight and not dependent on the protein charge.
NovexTM Precast Gel
The separation of proteoglycans is often carried out on Novex Tris-Glycine gels. The novex gels
do not contain SDS but it is included in the running and loading buffer. Use of SDS to mask the
charge in the protein molecule when separating proteoglycans, especially serglycin, may be
irrelevant. Proteoglycans are often highly negatively charged, due to the iduronic or glucuronic
acid and the substitution with sulphate in the GAG chains. Nevertheless, use of SDS seems to
prevent aggregation of proteoglycans, and should always be included in the loading and running
buffer.
Avoid high percent acrylamide gels when separating proteoglycans, since many proteoglycans do
not enter gels with acrylamide concentrations higher than 4 %. A 4 % gel on the other hand
separates smaller proteoglycans poorly. The best results are obtained with the 4-20 % gradient
gel used in this study.
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3 Methods
Reagents
2 x Loading buffer
10 x Running buffer
2.5 ml 0.5 M Tris-HCl (pH 6.8)
2.0 ml glycerol
4.0 ml 10 % (w/v) SDS
0.5 ml 0.1 % bromophenol blue
0.5 ml -mercaptoethanol
0.5 ml dH2O
29 g Tris-base
144 g glycine
10 g SDS
dH2O to 1 L
(pH adjustment not needed)
Protocol
1. Prepare 15 l samples in eppendorf tubes. Add 15 l 2 x loading buffer to each sample,
mix and centrifuge quickly. Preheat samples at 95 C for 5 minutes.
Samples with low salt concentration can be mixed: 20 l sample + 10 l sample buffer. Make sure that
there is no precipitation in sample after cooling to room temperature. This can be a problem, especially
with samples containing high salt concentrations (vacuum vaporised samples).
Use minimum 10000 cpm pr. sample, when using PhosphorImager to detect the radioactive signal.
2. Open the gel pouch and remove the gel cassette.
3. Wash the gel cassette with dH2O, pull out the comb and remove the tape at the bottom of
the gel.
4. Put the gel cassette in the Novex chamber with the well side facing the centre.
5. Fill the inner chamber with 1 x running buffer until 3 mm above the wells. Then fill the
outer chamber (total 500 ml buffer).
6. Wash the wells with 1 x running buffer, and load the samples into the wells (use 1 x
sample buffer in lanes without samples).
7. Connect the electrodes and turn on the power. Run the gel(s) at:
50 V (each gel) for 15 minutes, or until the samples have entered the gel.
100 V (each gel) for approximately 2 hours, or until the front line is 5 mm from the bottom.
8. Turn off the power, disconnect the electrodes and remove the gel(s).
9. Separate the two plates in the cassette by inserting a knife in the gap between the plates.
Preferentially remove the well side plate, allowing the gel to remain on the other.
10. Cut off the bottom lip with a scalpel, and wash the gel with dH2O.
11. Carefully press a Whatman paper on top of the gel while it is still wet, and transfer the gel
from the plate to the paper. Wet the paper with a few drops of dH2O if the gel does not
slip easily.
12. Place the gel in the Gel-dryer for 1 hour at 70 C.
13. Expose the gel in the PhosphorImager until next day. Do not cover the gel with plastic,
since plastic reduces the radioactive signal significantly.
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3 Methods
3.7.2 Anion-exchange chromatography
(Ion Exchange chromatography, Pharmacia Biotech)
Separation by ion-exchange chromatography depends on the reversible adsorption of charged
solute molecules to an immobilised ion exchange group of opposite charge. Positively charged
exchangers have negatively charged counter-ions (anions) available for exchange and are
therefore termed anion-exchangers. Ion-exchangers are divided into weak and strong. The terms
strong and weak refer to the extent of ionisation with variable pH and not the strength of binding.
In weak exchangers, the degree of dissociation and thus the exchange capacity varies much more
with pH.
DEAE Sephacel
DEAE sephacel is a weak anion-exchanger with the functional group diethylaminoethyl (DEAE)
linked to bead-formed cellulose. It is stable in aqueous solution between pH 2–12. DEAE
Sephacel is susceptible for microbial attack and should therefore be stored in the presence of
antimicrobial agents when not in use.
DEAE Sephacel can be used for buffer exchange or to purify and concentrate proteoglycans from
medium or cell lysate. Proteoglycans are immobilised to the column, while slightly negative,
neutral or positively charged molecules run through.
Tight plug
Buffer
DEAE Sephacel matrix
Peristaltic pump
Buffer
Figure 3-13: Anion exchange chromatography.
Reagents
Buffer A
20 mM Na2HPO4, pH 7.4
150 mM NaCl
Buffer C (elution buffer)
20 mM Na2HPO4, pH 7.4
1000 mM NaCl
Buffer B
20 mM Na2HPO4, pH 7.4
350 mM NaCl
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3 Methods
Procedure
1. Tighten the tip of a 5 ml syringe with glass wool, and make a 0,5 ml column. Remove the
rubber plug from the piston and make a small hole through the middle with a 1-200 l
pipette tip. Connect the tube coming from the peristaltic pump through the hole (Figure
3-13).
2. Equilibrate the DEAE-Sephacel column with 10 ml buffer A. To avoid drying of the
column, make sure that there is at least 0,5 ml buffer on top of the column.
3. Apply the medium to the column by pipetting on top, or pump it through the peristaltic
pump.
4. Wash with 5-10 ml buffer A, or until the colour from the medium is removed from the
column.
5. Wash with 10 ml buffer B.
6. Disconnect the peristaltic pump, and elute with 400 l buffer C in 4 steps. Collect the
eluate in separate eppendorf tubes. Most of the proteoglycans elute in the second fraction.
3.7.3 Sephadex G-50 Fine chromatography
Sephadex G-50 Fine is a gel filtration column with a separation range from 500 D to 10 kD
(globular proteins). Due to the low exclution limit, most macromolecules run outside the gel
beads, while small molecules enter the gel beads and are retained during gel filtration. This
property is used in a fast and easy gel filtration chromatographic method for removal of
unincorporated 35(S)sulphate together with buffer exchange in medium and cell lysate fractions
(Figure 3-14).
Add 1.5 ml
buffer
Apply 1.0 ml
sample
4 ml Sephadex
G-50 Fine gel
matrix
Let sample sink
into column
Let buffer sink
into column
Collecting
tube
Small ions are
retained in the
column
Eluted macromolecules
Figure 3-14: The Sephadex G-50 Fine technique for removal of unincorporated 35S(sulphate) and
buffer exchange of labelled macromolecules.
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3 Methods
Procedure
1. Suspend dry Sephadex G 50 Fine in a preferred buffer. Phosphate buffer (equilibration
buffer, page 77) is recommended if the samples are to be used with the Ni2+-affinity
system in further analysis.
2. Incubate the suspension at room temperature until next day, for swelling of the gel and
removal of air bubbles. (An alternative procedure is 2 hours incubation at 60 C followed
by cooling to room temperature before use.)
3. Cut a 10 ml pipette in half at the 7 ml mark, and discard the top. Add a small piece of
glass wool in the tip of the pipette, and fill it with swelled gel suspension until you got a 4
ml gel matrix.
4. Wash twice with 2 ml buffer (allow the buffer to sink by gravity force).
5. Apply 1 ml sample and allow it to sink by gravity force into the gel matrix.
6. Then add 1.5 ml buffer, and collect the run through.
7. Throw away the pipette with the gel matrix after use. Treat it as highly radioactive waste.
3.7.4 The FPLC system
The Fast Flow Liquid Chromatography (FPLC) system is a computer programmable
chromatography system delivered by Pharmacia Biotech. A computer unit controls two separate
pumps and a fraction collector (Figure 3-15). It can be programmed to control the total flow rate
through the column, and percent mixes of the two buffers if the pumps contain different buffers.
The fraction collector is programmed to separate the elute from the column into fraction of
optimal size. If wanted, it is also possible to include a conductivity meter and a
spectrophotometer unit.
All running parameters are programmed into a method file in an editor programme. This file is
then used by the computer system when running a sample. Both Superose 6 and Hi-Trap
chelating (Ni2+-affinity) columns are used with the FPLC system. The method files for these two
columns can be found at page 74 (Superose 6) and page 77 (Hi-Trap chelating).
Buffer A
Injector
Buffer B
Buffer mixer
Sample
loop
Column
Pump A
Pump B
1.0
0
Fraction collector
Control computer
Figure 3-15: The FPLC system.
71
3 Methods
72
3 Methods
3.7.5 Superose 6 column
(Superose® 6 HR 10/30 instruction manual)
Superose 6 is a gel filtration column with a separation range from 5 to 5000 kD. The high
exclusion limit is still too small for separation of the largest proteoglycans. Due to the large GAG
chains, many proteoglycans exclude the exceed limit of the column. However, serglycin secreted
from both U937 cells and transfected MDCK II cells have been found to elute after the void
volume.
Superose 6 columns can be used to determine the GAG chain length after -elimination and for
analysing the remaining intact PGs after degradation of GAGs on intact proteoglycans. The small
degradation products obtained by degrading of glycosaminoglycan chains from intact
proteoglycans will elute in the Vt. When 35(S)sulphate labelled proteoglycans are analysed, the
35
(S)sulphate will be incorporated into the GAG chains. Degradation of the GAG chains on the
core protein will thus be observed as a shift in the signal from the proteoglycan peak to the Vt
(Figure 3-16).
3000
c o nt ro l
Degradation
products
c A B C t re a t m e nt
Counts (cpm)
2500
H N O 2 t re a t m e nt
2000
Proteoglycan
fraction
1500
1000
500
0
-0,2
0,0
0,2
0,4
0,6
0,8
1,0
1,2
Kav - value
Figure 3-16: Superose 6 gel filtration of 35S(sulphate) labelled macromolecules.
A typical diagram after chondroitinase ABC lysase and HNO 2 treatment.
Reagents
Running buffer
20 mM Tris-HCl, pH 8,0
150 mM Sodium chloride
0,5 % Triton X-100
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3 Methods
Method file
The Superose 6 column is used with the FLPC system. The running programme contains the
following parameters.
0.00
1.00
13.00
56.00
70.00
FLOW
INJ_VALVE
FRACTION_COLLECTOR
FRACTION_COLLECTOR
END_METHOD
0.42
Inject
Start
Stop
{}
{tube 1}
{tube 35}
{end}
Procedure
1. Program the fraction collector with fraction size 1.25 and flow rate 45 (collect 0.5 ml
samples).
2. Prepare a sample containing at least 3000 cpm/200 l. Add 10x running buffer to 1x final
concentration. Make at least 30 l excess sample to avoid getting air bubbles when
injecting the sample into the loop.
Control the pH of the sample, and adjust to pH 7 - 8 after nitrous acid degradation or -elimination when
the samples is used on the Superose 6 column. At alkaline pH, proteins in the sample tend to denature when
the 10x running buffer is added, and the column starts packing after only a few samples.
3. Inject the sample (200 l loop) and start the programme. Injection of more than 200 l
sample will reduce the separation.
Column cleaning
Proteoglycans tends to tighten the column, which may cause high background pressure. As the
pressure increases, it enhances the problem by packing the Superose gel particles more tightly,
which again raises the background pressure. Superose 6 columns should not be used at pressures
above 1,5 MPa, and column cleaning is recommended if some of the underlying points are
observed.
 an increased back pressure
 a space that has become visible between adaptor and filter
 a colour change at the top of the column
 a loss of resolution
Procedure
Use a flow rate of 0.2 ml/ml during the cleaning procedure.
1. Wash with 25 ml 50 % acetic acid.
2. Wash with 25 ml water.
3. Wash with 20 % ethanol.
4. Wash with 25 ml water.
5. Wash with 25 ml 0.1 M NaOH.
6. Wash with 25 ml 4 M guanidine.
7. Rinse with 25 ml water and 3 x 200 l injections of 50 % acetic acid.
8. Equilibrate with buffer.
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3 Methods
3.8 Purification of serglycin-His-flag
3.8.1 Ni2+-affinity chromatography
(Ni-NTA spin kit, Qiagen)
(Hi-Trap chelating column user guide, Pharmacia Biotech)
Purification of His-flag molecules is based on the ability of histidines to form complexes with
transition metal ions, combined with a resin with high affinity for the same transition metal ion
(Figure 3-17). The high affinity of the resin for 6xHis-tagged proteins or peptides is due to both
the specificity of the interaction between histidine residues and immobilised transition metal
ions, and to the strength with which these ions are held to the resin.
R
NH
O
C=O
CH
CH2
N
OH
N
NH
N
Ni2+
O=C
N
CH
CH2
C
-O
CH2
C
-O
N
CH2
-O
O
CH2 CH2 CH2 CH
CH2 CH2 CH22 CH2 O
CH
C
O
NH
R
Figure 3-17: Binding of a histidine tag to a Ni2+ ion stabilised by a matrix.
In the end of a spacer arm is a Ni2+ ion stabilised by a quadradentate chelating group
(nitrilotriacetic acid). This group occupies four of six sites in the nickel coordination sphere, while
the two other sites are used in the binding to the imidazole rings in two histidines.
The metal ions most often used are Cu2+ and Ni2+ followed by other ions as Zn2+, Co2+ and Ca2+.
It is not possible to predict which ion is most suitable for the purification, detection or assay, but
the binding strengths are often Cu2+ > Ni2+ > Zn2+. Even though Cu2+ has the best binding
strength, it is not always the best choice. Stronger binding of the 6xHis tagged protein is also
followed by stronger unspecific binding. Besides, a strong binding may also need elution
conditions that denature the protein. Pilot experiments with different ions should be done if
optimal conditions are important.
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3 Methods
3.8.2 Hi-Trap chelating Ni2+-column
Several manufacturers provide material for purification, detection, and assay of 6xHis tagged
molecules. In this thesis, the Hi-Trap chelating column was used to purify serglycin-His-flag
secreted by the transfected MDCK II cells.
The Hi-Trap chelating column is a pre-packed, ready to use column made by Pharmacia Biotech,
which can be connected to the FPLC system. The chelating Sepharose High Performance resin
consists of highly cross-linked agarose beads, to which iminodiacetic acid has been coupled with
stable ether bounds via a seven atom spacer arm. Three of these iminodiacetic acid arms form a
tridendate with a transition metal ion in the center, and occupies three of six sites in the nickel
coordination sphere (Figure 3-18). Two other sites are used in the binding of two different, but
nearby histidines.
H2O
CO
O
CH2
N
Ni2+
H2O
O
H2O
CH2
CO
Figure 3-18: Binding of the Ni2+ ion to the matrix.
Three acetic groups stabilise the Ni2+ ion. Two of the
three free sites in the coordination sphere are used in the
binding to two histidines.
Choice of buffer
The native buffer system recommended for the His-TrapTM kit from Pharmacia Biotech was used
in the initial tests to optimise the purification procedure (20 mM phosphate buffer (pH 7.4)
containing 500 mM NaCl). Usually, when working with proteoglycans, the harvested medium is
diluted with guanidine until 4 M final concentration. However, this was not done, since
guanidine will interfere with the metal-chelating system. Another change compared to usual
buffer systems for chromatography of proteoglycans, is the removal of Tris-buffer, which also
will interfere with the metal-chelating system. Reagents with secondary or tertiary amines will
reduce the nickel ions, and thus reduce or even eliminate the specific binding between the bound
metal ion and the His-tag molecule. Other reagents that interfere with the ionic binding are
HEPES, MOPS, EDTA, EGTA, DTT, DTE, SDS, Gly, Gln, Asp, His, NH3.
The buffer system finally used with the Hi-Trap chelating column, contains less NaCl than
recommended in the His-TrapTM kit. A decrease in NaCl concentration to 150 mM instead of 500
mM did not seem to increase the unspecific binding of other proteoglycans to the column. High
salt concentration in the eluted material causes problems for later analysis, especially SDSPAGE, and thus it is preferred to keep it at a minimum.
76
3 Methods
Reagents
Equilibration buffer (pump A)
20 mM sodium phosphate pH 7.4
150 mM sodium chloride
1.42 g Na2HPO4
3.22 g NaCl
500 ml buffer
Elution buffer (pump B)
20 mM sodium phosphate pH 7.4
150 mM sodium chloride
100 mM imidazole
1.42 g Na2HPO4
3.22 g NaCl
3.40 g imidazole
500 ml buffer
Column stripping buffer
20 mM sodium phosphate pH 7.4
500 mM sodium chloride
50 mM EDTA
1.42 g Na2HPO4
14.61 g NaCl
25 ml 1 M EDTA
Ni2+ immobilisation buffer
20 mM sodium phosphate pH 7.4
100 mM Ni2+
1.42 g Na2HPO4
11.89 g NiCl2  6 H2O
500 ml buffer
500 ml buffer
FPLC programme for purification of serglycin-His-flag
2,00
100
90
Flow rate
Im idazole
1,50
80
70
1,25
60
1,00
50
0,75
40
30
0,50
20
0,25
Concentration (mM)
Flow rate (ml/min)
1,75
10
0,00
0
0
5
10
15
20
25
Figure 3-19: Illustration of the Hi-Trap chelating
programme.
Elute (ml)
Method file
0.00
0.00
0.00
0.00
2.00
8.00
8.00
15.00
15.00
21.00
21.00
24.00
35.00
INJ_VALVE
Inject
CONC_B
0.0
FLOW
0.25
FRACTION_COLLECTOR Start
FLOW
1.0
CONC_B
0.0
CONC_B
15.0
CONC_B
15.0
CONC_B
100.0
CONC_B
100.0
CONC_B
0.0
FRACTION_COLLECTOR Stop
END_METHOD
{slow flow (0.25 ml/min)}
{tube 1}
{normal flow (1 ml/min)}
{elution of low affinity}
{elution of high affinity}
{washing step}
{tube 30}
{end}
77
3 Methods
Procedure
Immobilisation of Ni2+ ions on the column
1. Inject 1000 l immobilisation buffer with flow rate 1.0 ml/min
2. Wash with 15 ml buffer A, then 10 ml buffer B. (The imidazole may remove loosely
bound Ni2+ ions.)
3. Remove the imidazole by washing with 10 ml buffer A.
Purification of serglycin-His-flag
1. Programme the fraction collector with fraction size 1.00 and flow rate 60. Use eppendorf
tubes when collecting. Some plastic tubes tend to bind proteoglycans to the tube walls.
2. Prepare 1100 l sample in an eppendorf tube.
3. Inject the sample into the loop (1000 l), and start the programme.
To facilitate formation of the specific binding, the flow rate starts at 0.25 ml/min and is then increased to
1.0 ml/min after 8 minutes, when the 1 ml sample has been injected into the column.
4. Count 10 % of each eluted fraction in scintillation counter.
Stripping of column (removal of Ni2+ ions)
1. Wash the column with 5-10 ml column stripping buffer.
2. Wash with 15 ml buffer A.
3. Equilibrate the column with 20 % ethanol.
Strip the column (remove Ni2+ ions) and equilibrate with 20 % ethanol prior to storage of the
column. It is possible to reuse the column several times, but the unspecific binding seems to
increase when the column is stripped several times. The unspecific binding is also suspected to
increase if the column is exposed to high pressure (personal observation). For that reason, do not
exceed the 1.0 ml/min flow rate.
78