ASC222
Animal Biotechnology
Laboratory manual
Autumn 2017
School of Animal and Veterinary Sciences,
Charles Sturt University
Written and Compiled by Dr. Nigel Urwin and Dr. Sameer Pant
1
Table of Contents
Exercise 1. Isolation of DNA from chicken blood and
assessment of its purity and concentration. ......................... 3
Exercise 2. Studying the effect of restriction endonucleases
on DNA by agarose gel electrophoresis. ............................. 11
Exercise 3. DNA amplification by Polymerase Chain
Reaction (PCR). ...................................................................... 7
Experiment 4. Restriction endonuclease digestion of a
plasmid and transformation of Escherichia coli with a
plasmid expressing green fluorescent protein.................... 11
Experiment 5. Purification of green fluorescent protein
from transformed Escherichia coli, assessment of purity
and estimation of its molecular weight. .............................. 27
Exercise 6. Detection of feline leukaemia virus (FeLV)
antigen using the enzyme linked immunosorbent assay
(ELISA). ................................................................................. 36
Exercise 7. Bioinformatics.................................................... 54
2
Exercise 1. Isolation of DNA from chicken blood and
assessment of its purity and concentration.
_____________________________________________________________________
Introduction
For studying genes and doing any sort of genetic manipulation at some stage we have
to extract nucleic acids from one sources or another. There are various methods and
many are very similar regardless of the source (animal, plant, or micro-organism).
Essentially a tissue is selected and with the exception of fluids the tissue is ground up
to release single cells or break cell walls. Sometimes enzymes are used to
enzymatically remove cell walls. The cells are then placed in a neutral buffer with
some salts such as guanidine hydrochloride which inhibit the action of enzymes
within the cells which can degrade nucleic acids and a detergent which lyses cellular
membranes including those of organelles like nuclei and mitochondria. The nucleic
acids are therefore released. Proteins are then usually digested with proteases and
precipitated by adding large amounts of salt and then the cell debris and proteins are
removed leaving nucleic acids in solution. In the method we will use today DNA
from detergent lysed cells is passed over a resin in a column. Under conditions of high
salt the DNA binds to the resin in the column. The column is then washed to remove
any remaining proteins and other contaminants and the pure DNA is finally eluted
from the column in a low salt buffer.
Chicken blood is a rich source of DNA since bird and some fish erythrocytes have
nuclei. We will extract DNA from just 15 µL of chicken blood following which we
will assess its’ yield, concentration and purity.
Methods
Extraction of DNA from chicken blood
1. You are supplied with a microcentrifuge containing 15 µL of chicken blood.
2. Add 15 µL of chicken blood to the 20 µL of proteinase K in the proteinase K
tube provided (e.g. exactly 20ul in the tube). Add 200 µL of phosphate
buffered saline solution (PBS). Mix until homogenous.
3. Add 200 µl of AL buffer. Mix thoroughly and immediately by vortexing and
incubate the sample at 56oC for 5 min. Caution: AL and AW1 buffer
contains guanidine hydrochloride which is a strong protein denaturant so if
you spill any on your skin or clothes wash off immediately with water. This
step lyses your cells releasing nucleic acids and you may notice an increase in
viscosity of the solution.
4. Add 200 µl of ethanol and mix by vortexing (check visually that the solutions
actually mix). Pipette this mixture into the DNeasy spin column placed in the
2 mL collection tube with the column. Centrifuge the column in the tube at
3
6000 x g (8000 rpm) for 1 min. Discard the flow through and the collection
tube. In this step your DNA is absorbed onto the column.
5. Place the DNeasy coumn in a new 2 mL tube and pipette 500 µL of AW1
buffer into the DNeasy column. Cap and centrifuge the column in the tube at
6000 x g (8000 rpm) for 1 min. Discard the flow through and the collection
tube.
6. Place the DNeasy column in a new 2 mL tube and pipette 500 µL of AW2
buffer into the DNeasy column. Cap and centrifuge the column in the tube at
6000 x g (8000 rpm) for 1 min. Discard the flow through place the DNeasy
column in the same collection tube.
7. Centrifuge the column for 3 min at 20000 x g (14000 rpm) to dry the
membrane in the column. Discard any flow through and the collection tube.
This step and the previous step wash your DNA on the column and remove
any remaining contaminating substances (eg. protein and carbohydrates).
8. Place the DNeasy spin column in a clean 1.5 mL microcentrifuge tube and add
100µL of AE buffer. Incubate at room temperature for 1 min and then
centrifuge for 1 min at 6000 x g (8000 rpm) to elute your DNA. In this step
your DNA comes off the column.
9. Your DNA is now in the microcentrifuge tube so cap the tube, label it with
your names and store on ice (DO NOT DISCARD THIS DNA SAMPLE AS
YOU WILL NEED IT NEXT WEEK). You can discard the DNeasy column.
Assessment of purity and concentration using a Nanodrop
spectrophotometer.
Nucleic acids absorb ultraviolet light due to the base ring structures in the double
helix. They absorb maximally at approximately 260 nm. The more concentrated your
DNA sample the higher the absorbance value. (Remember the Beer Lambert Law
from chemistry?). As a rule of thumb using a cuvette of 1cm path length (distance
through which light travels) an absorbance value of 1 at 260nm will be approximately
equal to 50 μg/mL for DNA.
Concentration of DNA = Absorbance at 260nm (of DNA solution in equivalent
of a 1cm pathlength)
x 50
DNA concentration =
μg/mL
Other molecules within cells can absorb ultraviolet wavelengths of light and a major
class of these is proteins due to ring structures of certain amino acids of which
proteins are made. Proteins absorb maximally at 280 nm. If we have removed all of
these then we should have a sample with low absorbance at 280 nm. In practice to get
an estimate of purity of our DNA sample we look at the ratio of absorbance at 260 nm
4
relative to that at 280nm (Absorbance 260nm/Absorbance 280nm). A pure DNA
sample will have a ratio of 1.7 or higher.
Absorbance 260nm/Absorbance 280nm =
In essence we assess the quality of the nucleic acid from the 260/280 ratio and the
concentration from its absorbance at 260nm. We will do this using a Nanodrop
spectrophotometer as follows
The ‘Nanodrop’ spectrophometer is like a conventional spectrophotometer but which
doesn’t require conventional cuvettes and uses volumes of <5μL of sample. The
instrument consists of a light source and detector in between which you place your
sample. The instrument can perform a ‘scan’ in which it measures the absorbance of
your solutions at different wavelengths for you. The instrument will be demonstrated
to you and zero’d on an AE buffer blank.
1. When you have your sample wipe the platform with a Kimwipe and pipette
3μLof your DNA solution on to the stage. It will be visible as a single drop.
Lower the lid onto the drop and press ‘measure’ sample.
2. Observe the shape of the graph and record the following data:
a. Absorbance at 260nm ______________________________________
b. Absorbance at 280nm_______________________________________
c. Concentration in ng/μL or μg/mL. _____________________________
3. When you have finished your sample wipe the drop off the stage ready for the
next user.
Questions
1. Calculate the concentration of your own DNA solution yourself based on the
absorbance at 260nm below and comment on if it is different from the
concentration the instrument calculated for you.
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
2. What possible contaminants might we have co-purified and what might be the
effects of the extraction procedure on our DNA?
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
5
3. How might we improve our extraction procedure to remove contaminants of
our DNA.
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
4. What properties of DNA did the experiment demonstrate?
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
5. Using the internet determine how much DNA you would expect to purify from
1 mL of undiluted chicken blood. Hint: to determine this you will need to
know how many cells per mL of chicken blood and the amount of DNA per
cell in micrograms. Show your working.
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
6
Exercise 2. Studying the effect of restriction
endonucleases on DNA by agarose gel
electrophoresis.
Introduction
The DNA of different organisms varies in base composition. We can determine
something of its nature by simply digesting it with restriction endonucleases and
observing the sizes of DNA fragments, which are generated by different enzymes.
Restriction endonucleases are enzymes isolated from bacteria that restrict the entry of
foreign DNA by cutting them across the double helix at very specific sequences. In
this experiment we will digest our chicken blood DNA sample, prepared previously,
with two enzymes, which cut at hexa-nucleotide sequences, and two enzymes, which
cut at tetra-nucleotide sequences. We will then separate the DNA fragments generated
using a technique called agarose gel electrophoresis. Electrophoresis is the movement
of charged particles in an electric field usually in a gel. In this case the gel is a slab of
agarose, a jelly like polymer extracted from seaweed. To prepare the gel the agarose
powder is weighed out and boiled up in buffer. It is poured in a mould and allowed to
cool and set (just like making a jelly at home! Aeroplane Jelly for me!). The gel is
submerged in a tank of buffer with electrodes at either end. The DNA samples are
placed in slots within the gel at one end. When a voltage is applied across the gel the
DNA fragments migrate towards the positive electrode through the gel matrix since
they are negatively charged due to the phosphate groups on the DNA backbone.
Because the movement of DNA fragments is retarded within the gel larger fragments
move less quickly than smaller ones and so we separate out the fragments on the basis
of size. DNA is not visible under normal light so we stain the gel with DNA specific
dyes which fluoresces under UV light (just like that white tee-shirt you wore at the
disco last week!). Dyes commonly used are ethidium bromide, SYBR green and Gel
Red. Ethidium bromide is a powerful mutagen so we will use either SYBR green or
Gel Red (or alternative ‘safe’ dye) as they are relatively non-toxic. The stain is
included in the gel for you. In addition to our test samples we separate a series of
DNA fragments of known size (standards) so we can estimate the size of our DNA
fragments generated. This is sometimes called a DNA ladder or molecular weight
marker set.
Source:http://course1.winona.edu/
kbates/Bio241/images/figure-17-02.jpg
7
Methods
1. Set up the following reactions in microfuge tubes in your ice bucket. Pipette
the reagents onto the side walls of the tubes and use a fresh tip for each one.
To mix just cap and spin the tube at full speed in a microcentrifuge for a
second or two. Note: Typically when we digest DNA with a restriction enzyme
we digest 1µg of DNA in a test tube in a final volume of 20 µL made up with
sterile distilled water. The reaction also contains a suitable buffer so the
enzyme is stable and the restriction enzyme of choice.
Reaction component
Tube Number
1
2
3
4
5
DNA volume for 1 μg (μL)
5
5
5
5
5
Sterile water volume (μL)
13
12
12
12
12
X 10 Buffer (μL)
2
2
2
2
2
BamHI (μL)
-
1
-
-
-
Sau3AI (μL)
-
-
1
-
-
MspI (μL)
-
-
-
1
-
HaeIII (μL)
-
-
-
-
1
2. Place all other tubes in a 37oC water bath and incubate for 30 min.
3. Remove tubes and add 5 μl of loading dye to each. Mix and spin as above and
place all tubes on ice until you are ready to load your gel.
4. Load your gel (the demonstrator will show you how) and record which slots
you placed your samples in. Make sure a set of DNA standards (DNA ladder)
are electrophoresed on your gel with your samples. After the electrophoresis is
completed and the gel is placed on a transilluminator (UV light box). Make
sure the Perspex shield in over the gel and UV light. When you have observed
the gel it will be photographed for you.
8
Questions
1. What size is your chicken blood DNA? How big would you expect it to be and
are their any bands in the sample which were unexpected and what are they?
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
2. What are the size ranges of DNA fragments produced by digestion with
a)
b)
c)
d)
BamHI
Sau3AI
MspI
HaeIII
3. What are the cleavage sites for all four enzymes? What do you notice about
them, if anything?
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
4. Would you expect digestion with BamHI or Sau3AI to give you fragments of
the same size or different and why?
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
5. Would you expect all target sites for Sau3AI to be digested by BamHI ….Yes
or No and why or why not?
_______________________________________________________________
_______________________________________________________________
6. Would you expect all target sites for BamHI to be digested by Sau3AI ….Yes
or No and why or why not?
_______________________________________________________________
_______________________________________________________________
9
7. Would you expect digestion with MspI or HaeIII to give you fragments of the
same size or different and why?
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
8. If the size range of fragments is different for the two enzymes in 5. Then can
you offer an explanation as to why this might be?
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
10
Exercise 3. DNA amplification by polymerase Chain
Reaction (PCR)
Introduction
The human genome is made up of approximately 3 billion chemical base pairs.
Scientists often need to isolate a very specific segment of DNA from within a vast
amount of genetic material. Since this segment is just one tiny piece of the genome,
they need many copies to have enough to work with.
The polymerase chain reaction (PCR) is a method used by scientists to rapidly copy,
in vitro, specific segments of DNA. By mimicking some of the DNA replication
strategies employed by living cells, PCR has the capacity for churning out millions of
copies of a particular DNA region. It has found use in forensic science, in the
diagnosis of genetic disease, and in the cloning of rare genes. One of the reasons PCR
has become such a popular technique is that it does not require much starting material.
PCR can be used to amplify DNA recovered from a plucked hair, from a small spot of
blood, or from the back of a licked postage stamp.
There are some essential reaction components and conditions needed to amplify DNA
by PCR. First and foremost, it is necessary to have a sample of DNA containing the
segment you wish to amplify. This DNA is called the template because it provides
the pattern of base sequence to be duplicated during the PCR process. Along with
template DNA, PCR requires two short single-stranded pieces of DNA called
primers. These are usually about 20 bases in length and are complementary to
opposite strands of the template at the ends of the target DNA segment being
amplified. Primers attach (anneal) to their complementary sites on the template and
are used as initiation sites for synthesis of new DNA strands. Deoxynucleoside
triphosphates containing the bases A, C, G, and T (NTPs) are also added to the
reaction. The enzyme DNA polymerase binds to one end of each annealed primer and
strings the deoxynucleotides together to form new DNA chains complementary to the
template. The DNA polymerase enzyme requires the metal ion magnesium (Mg++)
for its activity. It is supplied to the reaction in the form of MgCl2 salt. A buffer is
used to maintain an optimal pH level for the DNA polymerase reaction.
PCR is accomplished by cycling a reaction through several temperature steps. In the
first step, the two strands of the template DNA molecule are separated, or denatured,
by exposure to a high temperature (usually 94° to 96°C). Once in a single-stranded
form, the bases of the template DNA are exposed and are free to interact with the
primers. In the second step of PCR, called annealing, the reaction is brought down to
a temperature usually between 37 ̊C to 65 ̊C. At this lower temperature, stable
hydrogen bonds can form between the complementary bases of the primers and
template. Although human genomic DNA is billions of base pairs in length, the
primers require only seconds to locate and anneal to their complementary sites. In the
third step of PCR, called extension, the reaction temperature is raised to an
intermediate level (65 ̊C to 72 ̊C). During this step, the DNA polymerase starts adding
nucleotides to the ends of the annealed primers. These three phases are repeated over
and over again, doubling the number of DNA molecules with each cycle. After 25 to
40 cycles, millions of copies of target DNA are produced. The PCR process taken
through four cycles is illustrated on the following page (Figure 1).
11
Figure 1. The first four cycles of the polymerase chain reaction
12
In the following laboratory exercise, you will use PCR to amplify a replicationassociated protein gene (designated as “Rep”) of a small virus that causes the beak
and feather disease (BFDV). Beak and feather disease is a common viral disease of
wild psittacine birds worldwide that is characterized by chronic, progressive,
symmetrical feather dystrophy and occasional beak deformity. You will be provided
with a positive control sample, a negative control sample, and a test sample. After you
finish PCR, you will determine whether the test sample is positive for BFDV by
separating the DNA in your PCR sample on an agarose gel via electrophoresis, a
process that separates DNA by size.
Required reagents:
•
•
•
•
PCR Master Mix, containing: deoxyribonucleotide triphosphates (dNTPs),
Mg2+ and Taq DNA Polymerase
Deionised H20
Primer Mix
Template DNA
Equipment & Supplies
•
•
PCR tube
1-20 μl pipettes and tips
Shared Items
•
•
Vortex
PCR machine
13
Student Procedure
Student Procedure
1. Obtain a 200 μl or 500 μl PCR tube and label it with a unique ID
Note: Keep PCR tube on ice when setting up the reaction.
2. Pipet 20 μl of Master Mix into your PCR tube
3.
Change your pipet tip and add 20 μL of Primer Mix into your
PCR tube
Forward Primer sequence 5´-TGCAAGGCTACTTTCATTTTA-3´
Reverse Primer sequence 5´-TCCTTCATTTTGCGTCC-3´
4. With a new pipet tip, add 10 μL of the test, positive control and
negative control samples into the three PCR tubes, and label these
as (T), (+C) and (-C) respectively.
What is the total volume in each tube? _______ μL
Note: Make sure that all the liquids are settled into the bottom of
the tube and not on the side of the tube or in the cap. If not, you
can give the tube a quick spin in the centrifuge. Do not pipette up
and down; it introduces error.
6. Check the volume of your PCR tube by comparing it to a reference
PCR tube with 50 μL in it. It should be near the thermal cycler, set by
your demonstrators.
7. Place your reaction into the thermal cycler and record the location
of your tube on the grid (e.g.R1C10 – rows being counted from left to
right and columns from top to bottom)
8. The cycling protocol for amplification of Rep gene of BFDV:
1) 95°C hold for 2 minutes
2) 30 cycles of:
95°C for 30 seconds
57°C for 30 seconds
72°C for 30 seconds
3) 72°C hold for 10 minutes
4) 4°C hold, ∞ infinity
14
Post-Lab Focus Questions
1. Draw out a pictorial representation of PCR. Include such steps as the separation
of DNA strands, binding of primers, and elongation.
15
2.
a. Suppose you begin a PCR reaction with 1 piece of double stranded DNA.
After 28 cycles of replication, how many pieces of double stranded DNA do you
now have?
b. At the end of the entire PCR reaction, are the original DNA strands bound to
each other or to new DNA strands? Explain.
3.
What exactly is PCR used for and why is it an effective and important technique?
4.
What is the role of the DNA primers in PCR?
5.
What do you think would happen if you added the wrong primers? Explain.
6.
What do you think would happen if you added only 3 out of the 4 total types of
dNTPs? Explain.
16
Experiment 4. Restriction endonuclease digestion of a
plasmid and transformation of Escherichia coli with a
plasmid expressing green fluorescent protein.
Introduction
This experiment will be done in two parts:
A.
Restriction endonuclease digestion of pGLO with EcoRV and EcoRI
B.
Transformation of E. coli with pGLO and induction of GFP expression.
Restriction endonucleases are enzymes that recognise and cut specific sequences in
DNA. They are very important tools in molecular biology. The recognition
sequences of over one hundred of these enzymes are known and thus enzymes can be
selected to cleave DNA precisely at desired sequences. Once DNA has been cut,
selected fragments can be joined together (ligation) to form recombinant DNA.
Plasmids are circular double stranded molecules of DNA. They are commonly found
in bacteria and range in size from 2000 nucleotides to tens of thousands of nucleotides
in length. Many copies of plasmids can exist in a single bacterial cell whereas only a
single chromosome exists per cell. Plasmids undergo replication similar to that of the
chromosomal DNA and are carried from one generation to the next.
Plasmids such as pBR322, pUC18 and pGLO are used to transfer DNA from any
organism into the bacterium E. coli. From here genes can be modified and then
transferred to other organisms like animals, plants and fungi. Plasmids used to do this
are often called ‘vectors’. They can easily be extracted from bacterial cultures, then
genetically modified, to include genes of our choice and then transferred back to
bacteria and thence to other organisms. In the first part of this exercise we will digest
the plasmid pGLO (figure 1.) with two restriction enzymes, EcoRV and EcoRI in
reactions containing each individual enzyme and in reactions containing both
enzymes. We will observe and determine the number and sizes of the fragments of
DNA generated after separating them by agarose gel electrophoresis. We will attempt
to derive a simple restriction enzyme map of this plasmid for the two enzymes.
17
Figure 1: Plasmid pGLO The arrows indicate protein coding region
Source:
http://www.med.hokudai.ac.jp/~bio-1w/jisshuu/matome03/main.html
In the second part of this exercise we will examine a technique used to transfer a
plasmid to the bacterium E. coli. This process is called transformation and involves
uptake of naked DNA (our plasmid) by cells in solution. Although transformation
occurs naturally in nature, in the laboratory we increase the permeability of our cells
to DNA by treating them with a solution of calcium chloride. This increases the
efficiency of the process. We will then select cells containing our plasmid by virtue of
a trait encoded by the ‘bla’ gene on our plasmid DNA which encodes resistance to
the antibiotic ampicillin. In addition the plasmid we will use contains a gene
encoding Green Fluorescent Protein (GFP). GFP originally came from a jelly fish
Aequorea victoria. GFP is a protein which fluoresces bright green under UV light and
the gene has been taken from the jelly fish and placed in the plasmid pGLO. The gene
is only transcribed in the presence of the sugar inducer arabinose so that we can
control its expression. We will observe how the concentration of this sugar affects
expression of the GFP gene.
18
Figure 2.: Plasmid encoded proteins produced by a bacterial cell under conditions which induce GFP expression
Source: Bio-Rad GFP protein purification kit manual
Method
Restriction endonuclease digestion of pGLO
1.
Using the reagents provided in your ice bucket and the plasmid pGLO at a
concentration of 0.2 µg/µl (TUBE A) set up the following reactions in
microfuge tubes. You can pipette the reagents on to the side walls of the tubes
on ice.
Restriction enzyme digests:
Reagent volumes
(added in this order)
Tube 1
Tube 2
Tube 3
Tube 4
Buffer (x10)
2 µl
2 µl
2 µl
2 µl
pGLO (0.2 µg/µl)
4 µl
4µl
4µl
4µl
12 µl
12 µl
10 µl
14 µl
2 µl
-
2 µl
--
-
2 µl
2 µl
-
20 µl
20 µl
20 µl
20 µl
Sterile water
EcoRV (5U/µl)
EcoRI (5U/µl)
Total volume
2.
To make sure all the contents are at the bottom of the tubes and mixed give the
tubes a quick spin in the microcentrifuge at 13000 rpm for 5 secs.
19
3.
Incubate your tubes at 37oC for 1 hour (proceed to Section B during this time).
4.
When the incubation is finished, add 5 µl of loading dye solution to each tube,
mix gently and re-spin again to bring contents to the bottom of the tubes.
5.
Load your samples on to the agarose gel as shown by your demonstrator and
record which gel and what lanes your samples are in. There will be lanes
containing molecular weight marker DNA’s loaded for you by the demonstrator.
Record these as well.
The gels we will use in this exercise are called ‘E-gels’. These are agarose gels
without external buffer reservoirs and electrodes which make contact with the
gel itself. The dye which binds to DNA and fluoresces under UV/ blue light is
included in the gel and the apparatus sits on its own light box so that we can
visualise the electrophoresis as it occurs. The separation is extremely fast (710min) compared to conventional agarose gels (30 mins to 1 hour). After
electrophoresis the gels can be removed and photographed so that we can
visualise and record them for you.
7.
Measure the distance moved by each of the bands in your digests and the
distance moved by each of the molecular weight marker bands on your gel from
the well and record below.
8.
Plot a graph (standard curve) of log molecular weight (Y-axis) against distance
moved to your each of the marker bands (X-axis). Use the standard curve to
determine the molecular weight of your bands in your digests. If you do not
know how to do this speak to your demonstrator or lecturer. From these deduce
the size of pGLO and draw a circular map of the plasmid with approximate
locations of enzyme sites and sizes of fragments.
20
Sizes of
bands in
basepairs
in the
DNA
ladder
(bp)
Log size of
bands in
basepairs in
the DNA
ladder
(bp)
Distance
moved from
origin by
bands in
DNA ladder
(mm)
Distance moved from origin by bands in digests (mm)
EcoRI
EcoRV
EcoRI
+ EcoRV
Undigested DNA
Include the picture of your gel and graph in this report.
CAUTION: DNA binding dyes like SYBR green and Gel Red whilst ‘safe’ still
requires the use of gloves and disposal of these and the gels in the containers
provided. These and many reagents have not been fully tested on animals so
their toxicity to some extent is unknown.
21
Transformation of E. coli with pGLO and selection for ampicillin
resistance and GFP expression.
You are provided with a preparation of E. coli (‘Competent E. coli cells’) that have
been treated with CaCl2 in such a way that when combined with pGLO in solution the
plasmid will move through the cell membrane and into the bacteria, i.e. the bacteria
will be transformed.
Caution:
Dispose of all rubbish in the autoclave bags provided.
Set up the bacterial transformation as follows: work in pairs
(Note: keep the competent cells on ice at all times)
1.
Add 5 µL (5 ng) of pGLO (1 ng/µL ) to a fresh microfuge tube - label the tube
‘+ pGLO’. Note use plasmid in ‘TUBE B’.
2.
Add 5 µL of sterile distilled water to another microfuge tube - label ‘ - pGLO’.
3.
Place the tubes on ice and add 100 µL of Competent E. coli cells to each tube.
4.
Leave on ice for 10 minutes then place the tube in a 42oC water bath for exactly
90 seconds.
5.
Put on ice for 2 minutes then add 1 ml of L-broth.
6.
Incubate at 37oC for 5-10 minutes (during this time finish off Section A).
7.
Remove from an aliquot of 100 µl from the ‘+ pGLO’ tube, place on an L-agar
plate containing amipicillin (+ amp plate) and spread over the entire plate as
demonstrated by your demonstrator, aseptically. Place the second 100 µl
aliquot on a plate without ampicillin plate (-amp).
8.
Repeat step (7.) with cells from the –pGLO tube. You should have 4 plates in
total as follows - (label them):
a
b
c
d
+ pGLO +amp
- pGLO +amp
+ pGLO -amp
- pGLO -amp
9.
Remove from the ‘+ pGLO’ tube 3 x 100 µl aliquots and place each of them on
an L-agar plate containing ampicillin (+ amp plate) and arabinose (+ara) at
concentrations of 5 mg/mL, 1 mg/mL and 0.2 mg/mL and spread as before. In
total you should have 7 plates. These will be incubated at 32oC overnight and
we will examine them next week.
10.
Observe your plates and count colonies on all of them. Record your results in
Table 1 below.
22
Table 1.
Antibiotic
presence
-amp
+amp
-amp
+amp
+amp
+amp
+amp
Arabinose
concentration
(mg/mL)
0
0
0
0
5
1
0.2
Plasmid presence
in transformation
No of
colonies
Colour of colonies under
UV light
-pGLO
-pGLO
+pGLO
+pGLO
+pGLO
+pGLO
+pGLO
Calculation of transformation efficiency
Transformation procedures are often used to obtain recombinant DNA clones.
Transformation procedures are often used to obtain recombinant DNA clones. Using
fragments of DNA from some organism of interest which has been digested with a
restriction enzyme and ligated into a vector cut with the same enzyme the object of
the transformation is to recover a many transformants as possible, each with a
different fragment in it. In this way we can obtain a ‘library’ of many DNA
fragments. This strategy is called ‘Shotgun’ cloning and the number of individual
clones we obtain is critical to getting a representative library (i.e. clones with
fragments of every part of the genome). Here the more transformants we get the
better and this is described as transformation efficiency. Transformation efficiency is
defined as ‘the number of transformants per microgram of DNA’ used regardless of
number of cells and amount of DNA used in a single transformation.
You used 5 ng of DNA in your transformations so calculate the efficiency of your
technique. The best techniques will give you 1-5x109 transformants /µg and average
ones will give you 1-5x 105 transformants /µg. Note there is a difference of four
orders of magnitude here (10,000 fold). Record your result below.
Transformation efficiency =
transformants /µg
23
Questions
1. How many sites for each of your restriction enzymes are there in pGLO? What
is your estimate of the total size of this plasmid? What is the actual size of this
plasmid? Can you derive a restriction map for pGLO for the two enzymes
based on your data of fragment sizes in each of the digests? Draw it below and
label distances between the sites.
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2. How many bands were observed in the undigested pGLO? Why?
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3. If we wanted to clone a DNA fragment into this plasmid would either of the
restriction enzymes be suitable to digest the vector with? Explain your answer.
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4. Why did no colonies grow on the L-agar ampicillin plate from the
transformation with no pGLO?
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24
________________________________________________________________
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5. Consider the number of colonies from the transformation +pGLO when plated
on L-agar containing ampicillin compared to lacking ampicillin, what does
this say about the process of transformation?
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6. What is the mechanism of ampicillin resistance?
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7. Under what conditions did you observe expression of GFP? Was there a dose
dependent effect of arabinose on GFP expression?
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8. How does arabinose induce GFP expression in pGLO and what is araC in
figure 1?
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25
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9. How might we further increase expression of this gene in E. coli?
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26
Experiment 5. Purification of green fluorescent protein
from transformed Escherichia coli, assessment of
purity and estimation of its molecular weight.
Introduction
Biotechnology has revolutionised the way we produce many therapeutic proteins of
commercial value which used to be extracted from human or animal sources. These
include insulin which used to be extracted from pig pancrease, growth hormones and
interferon. These are now produced in genetically engineered micro-organisms like E.
coli or yeast or in mammalian cells grown in cultures in large fermentors. Following
growth of the cells expressing the desired protein product, the protein is then extracted
from the cells or culture media into which it is secreted. The protein must usually
remain functional during and following purification and no contaminating proteins
should be present in the final product.
In the previous experiment you genetically transformed E. coli with a plasmid which
causes expression of GFP in E. coli. Whilst GFP is not a commercially valuable
protein, in this experiment we will purify the GFP by column chromatography to
illustrate the principle. The presence of green fluorescent protein can be easily
monitored using a long wave UV light to monitor its fluorescence.
This exercise is in two parts. In the first we will inoculate a single colony of our GFP
expressing bacteria from the LB agar plates containing ampicillin and arabinose into a
small liquid culture and grow this overnight at 32oC. We will do the same with
another single colony from the same plate but place this one in a liquid culture which
will have only ampicillin in it and not arabinose. The cultures should be grown
optimally at 32oC and with shaking to enhance oxygen supply to the cells. This
enhances GFP production and correct folding and stability.
After overnight culture we will centrifuge cells to pellet them, break them open to
release the GFP and purify the GFP by column chromatography using a hydrophobic
interaction matrix. GFP has several stretches of hydrophobic amino acids. Normally
in solution these are in the interior of the protein away from the surface and water
molecules. Addition of salts to the protein mixture causes some proteins to precipitate
and in the case of GFP it changes its shape to expose hydrophobic regions. If the
bacterial proteins are passed over a column containing beads with hydrophobic
surfaces (hydrophobic interaction matrix) the GFP sticks to the beads whereas other
proteins do not and will flow straight through. The GFP can be removed from the
column by addition of a small quantity of low salt buffer. When we reduce the salt
concentration the GFP becomes less hydrophobic and detaches from the column and
can be collected in a purer form. The idea in purification of proteins is to remove
everything but the desired product in this case GFP.
In the second part of the exercise we will use a technique called polyacrylamide gel
electrophoresis (PAGE) to monitor product purity and determine the molecular weight
of our GFP.
27
We have already used electrophoresis to separate DNA fragments on the basis of size
in agarose gels. Many techniques use this principle in one form or another to separate
other biological molecules. Polyacrylamide gel electrophoresis (PAGE) of proteins is
one such technique in which proteins are separated on the basis of size, charge and
shape. Polyacrylamide is a polymer of a synthetic organic compound called
‘acrylamide’. It forms a gel which is analogous to a sponge. Proteins can be charged
by virtue of their side chains and if placed at one end of a slab of polyacrylamide will
move if a electrical charge is placed over the gel. Proteins will travel towards a
positive electrode if they are negatively charged and will do so at a rate determined by
their size and charge and shape. A technique in which proteins are separated based on
the above properties is called ‘Native-PAGE’ because the proteins are in their native
form. This technique is often used to separate proteins and give an indication of their
native molecular weights.
A variation on this technique uses a detergent called sodium dodecyl sulphate (SDS)
to denature the proteins. Prior to electrophoresis the proteins are treated with this
detergent which binds uniformly along the length of the polypeptide chains. It also
breaks secondary, tertiary and quaternary structure so subunits are separated. SDS is
negatively charged so the proteins have a negative charge proportional to their size.
When these are separated by SDS-PAGE they move according to their size alone.
This technique is very useful for estimating subunit molecular weight.
In SDS-PAGE the gels consist of two sections. The first is the ‘resolving gel’ which
has a high concentration of acrylamide and does the separating. The second region of
the gel in which the samples are loaded is called the ‘stacking gel’. This portion has a
low concentration of acrylamide and a slightly different buffer so proteins move
through this very quickly and it is used to concentrate them before they reach the
resolving portion of the gel.
28
To estimate molecular weight of native or denatured proteins we also
electrophoretically separate a mixture of proteins of known molecular weights
(standards) on the same gel. If we graph the distance moved by the protein standards
versus the log of their molecular weights we get a standard curve. The molecular
weight of unknowns can then be estimated from the distances they migrate in the gel
using the standard curve.
Most proteins are colourless and therefore to visualise them we stain the gel after
electrophoresis with a dye which will bind to proteins. Coomassie brilliant blue is
such a dye.
Figure 2: Polyacrylamide gel electrophoresis
We will sample the cell extracts before purification and after and electrophorese these
to visualise the purity of the crude extract and purified preparations and estimate the
molecular weight of the purified GFP
Methods
Part 1. - Growth of E. coli, extraction and purification of GFP by
Hydrophobic Interaction Chromatography (HIC).
Growth of E. coli in liquid culture.
1. From your plate containing the green fluorescent colonies and using an
inoculating loop pick a single colony aseptically and transfer it to 2ml of LB
containing ampicillin and arabinose (LB/amp/ara) in the 15 mL tube provided
marked ‘+ ara’. Twirl the loop to distribute the cells from the loop into the
medium. Flame your loop and repeat using another colony from the same plate
and transfer it to the tube labelled ‘- ara‘. This tube contains LB/amp media
without arabinose. Label with your name and the date and these tubes will be
incubated at 32oC for 24hrs in an orbital shaker for you. These culture may
have been set up for you and you will be provided with the cultures
aready grown.
29
Preparation of bacterial lysate and chromatography column
1. Observe your culture tubes under normal light and the UV light and note any
differences. Take all of your cells from the + ara tube and transfer to a 2ml
microcentrifuge tube using a Pasteur pipette. Repeat for the – ara tube. Cap the
tubes and centrifuge for 5 minutes at full speed.
2. Observe the tubes under UV light, pour off the supernatant and observe the
pellets again. Using graduated Pasteur pipettes transfer 250μL of TE solution
to each of your tubes and resuspend your cell pellets by vigorous pipetting up
and down or using the vortex mixer.
3. Using a clean pipette transfer 1 drop of lysozyme solution to your cells. Cap
the tubes and mix the contents by flicking the tube with your finger. Lysozyme
is a protein found in egg white which digests peptidoglycan, a major polymer
in bacterial cell walls. Incubate at room temperature for 5 mins.
4. Place your tubes in the ice bucket containing liquid nitrogen. You must wear
gloves and safety glasses and the tongs provided. Liquid nitrogen temperature
is -196oC so don’t get your hands in it. Leave in for 20 seconds and remove.
Place on ice for 30 seconds and thaw in your hand. This step lyses the bacteria
releasing the contents.
5. Place your tube in a centrifuge and pellet the bacterial debris by spinning for
10mins at full speed. Label a clean microfuge tube with your name and set
aside. Whilst your tube is spinning prepare your chromatography column as
follows.
6. Shake the column to mix the beads and shake down to get the beads down to
the bottom. Tapping the column base on the bench in vertical position will
help. Remove the top cap and snap off the bottom and allow all the liquid to
drain (takes about 3-5 mins). Support the column in the clamp and stand whilst
doing this.
7. Add 2ml of Equilibration buffer to the column and allow it to drain through
until no more buffer flow through the column. Cap the bottom of the column
to prevent further drainage.
8. After your centrifuge has stopped remove your tube and observe the
supernatant and pellet under UV light and record your observation. At this
point take 20μL samples of supernatant from both tubes ( +ara and – ara)
place them in screw capped microfuge tubes. Add 20 ul of sample buffer to
both tubes. Mix, cap them and place on ice for electrophoretic analysis next
week.
9. Transfer the remainder of the of supernatant of the +ara tube (250μL) to a
fresh tube taking care not to disturb the pellet. Add 250μL of binding buffer
30
to this tube, cap and mix. This tube contains GFP we will purify using the
column.
Protein chromatography
Figure 1. General strategy for purifying GFP by hydrophobic interaction chromatography
1. Label 3 collection tubes 1-3 and place in a rack. Remove the caps from the
bottom of your column and place in tube 1.
2. Transfer using a fresh Pasteur pipette all your supernatant plus binding buffer
to the top of the column in the centre just above the upper surface of the beads.
Allow the solution to drip through the column and collect what come out
(eluate) in tube 1.
3. After the column stops dripping transfer it to the second collection tube (2)
and pipette in 250μL of wash buffer onto the column and let the entire volume
flow through as before and collect it in tube 2. Examine the column and tubes
under UV light. Where is the GFP? Record your observations.
4. After the column stops dripping transfer it to the third collection tube and
pipette in 750μL of EB buffer and let the entire column volume flow through.
Whilst this is happening observe the column under UV light. Do not collect all
of the 750μL of eluate but only the few drops (2 or 3) which contain the GFP.
You will need to constantly monitor the column with the UV lamp to see
where it is so you don’t miss it eluting from the column. Collect the drops
containing GFP in tube 3 and the rest of the eluate (with no GFP) in another
tube.
5. Label 3 screw capped tubes BB, WB and EB and pipette 20μL of your flow
through from collection tube 1 into the tube labelled BB, 20μL of your flow
through from collection tube 2 into WB and 20μL of your flow through from
collection tube 3 into the tube labelled EB. Add 20ul of sample buffer (blue)
to all three tubes. Cap, mix and set aside (with the samples from step 8) for gel
31
analysis next week. Do not discard these tubes and put in the rack
provided.
Gel Electrophoresis
Checklist of material saved from previous experiment
You should have 5 tubes in total. The two labelled +ara and –ara should contain
samples of crude bacterial lysates one of which should have large quantities of GFP
and the other should have very little GFP. The remaining three tubes contain 3
samples of buffers which flowed through your column at various stages of our
procedure to purify GFP.
E. coli cells are made up of over a thousand different proteins and in purifying our
GFP we attempted to remove all of these apart from the recombinant protein. In this
practical we will separate the proteins in our samples on the basis of size and estimate
how many are present in the lysates and purified samples. We will also try to
determine the molecular weight of our recombinant protein GFP and assess its purity.
1. Place the tubes in a boiling water bath for 5 mins and allow them to cool to
room temperature and load up to 30 μl of each sample in a well on your gel.
Load 20 μl of the protein standards provided in a separate well.
2. The PAGE apparatus will be demonstrated to you but you will load your
samples. Make sure you have recorded which gel and which lane your load
your samples and in what order.
3. Fix on the top electrode and run the gel at 200 volts for approximately 30mins
- 1 hour or until the tracking dye in the sample buffer is ¾ of the length of the
gel.
4. Switch off the gel and remove the gel from the apparatus. Gently prise apart
the glass plates and the gel will remain attached to one plate. Observe your gel
under UV light and record whether you can see GFP or not and in which lanes
and samples.
5. Place the glass plate with the gel attached into the gel staining solution in the
sandwich box provided. Gently slide the gel off the glass plate into the
staining solution. Gently agitate the gel in staining solution for 10-15 mins by
gently rocking back and forth.
6. Pour off the gel stain and replace with destaining solution. Agitate the gel
gently in the solution and replace after 5 mins with fresh destain. After another
5 mins observe your gel on the light box provided and observe the bands of
protein in it. Your gel may need to be in destain overnight.
7. When destained the gels will be photographed for you. Measure the distance
moved by each protein standard and by the major protein(s) in your samples
Record in Table 1.
32
*NOTE: If your gels do not de-stain sufficiently for you to observe bands allow them
to de-stain overnight. We will photograph them for you and the photo should be
included in your report.
33
Questions
Part 1 – Purification of GFP
1. Describe the principle of HIC, name a type of resin used for this sort of
chromatography and define the chemical groups involved.
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2. Draw a brief flow diagram of the purification procedure below indicating at in
which step GFP is removed from the column.
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3. Find a diagram of GFP structure and include it in your report. Briefly describe
it’s overall structure.
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Part 2. - Assessment of purity of GFP and estimation of its molecular
weight by denaturing PAGE.
1. How many proteins do you estimate are present in E. coli from the number of
bands observed? Can we see them all and if not why not? How many proteins
is the E. coli genome estimated to encode?
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34
2. Can you see a protein band corresponding to GFP and what is its molecular
weight (you can determine this from the molecular weight standards).
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3. In what samples is GFP seen and why?
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4. Is your sample of ‘pure GFP’ actually pure and how do you tell? If this was a
therapeutic protein for use in humans to be administered by intravenous
injection what could be the possible consequences if the protein were not
pure?
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35
Exercise 6. Detection of feline leukaemia virus (FeLV)
antigen using the enzyme linked
immunosorbent assay (ELISA).
General Introduction to This ELISA Kit
The following section briefly describes the technical and conceptual points that are
directly related to the laboratory activities in this curriculum. Student understanding
of these points is extremely important to a successful outcome.
Microplate strips: Microplates are made of polystyrene which adsorbs (binds)
proteins by hydrophobic interaction. The plates provided in this kit have 96 wells,
arranged in 8 removeable rows of 12-well strips. Two students share one strip. Each
well holds approximately 250 microliters (μl).
Antigen: In this kit, the antigen is chicken gamma-globulin (purified from egg yolks)
which serves as a generic representative of any hypothetical antigen, protein or
otherwise.
Incubation times: The rate of binding depends on the incubation temperature and the
concentrations of the reagents. This kit has been optimized so that each incubation can
be performed for 5 minutes at room temperature. Exceeding this time or temperature
will cause an increase in color intensity and possibly some background color in the
negative controls.
Blocking: Blocking agents are added after antigen adsorption to prevent nonspecific
binding of antibodies to the plastic, which would produce false positive results. The
blocking agent may be a protein or a detergent (or both). Common blocking agents
include Tween 20 (a nonionic detergent that is used in this kit), nonfat dry milk,
gelatin, and bovine serum albumin (BSA). Although Tween 20 is a sufficient block
for this protocol, you may wish to add the following blocking step for teaching
purposes: have the students add 50 μl of 1% gelatin in wash buffer to their wells for
15 min after the addition of the antigen and then perform a wash step.
Primary (1°) antibodies: The antibodies that recognize and bind to the antigen in an
immunoassay are primary antibodies. In this kit, the primary antibody is a polyclonal
rabbit antibody raised against chicken gamma-globulin. In the ELISA antibody test
starting on page 47, this primary antibody simulates human antibodies in a sample of
human serum.
Secondary (2°) antibodies: Secondary antibodies recognize and bind to primary
antibodies. They are made in animals of a different species than that used to make the
primary antibody. For this kit, goats were immunized with rabbit IgG to make the
secondary antibodies.
Colourimetric detection: Secondary antibodies for ELISA are linked to enzymes.
36
Detection of secondary antibodies that are bound to primary antibodies occurs by an
enzyme-substrate reaction. In this kit, the secondary antibody is linked to horseradish
peroxidase (HRP). In the presence of hydrogen peroxide (H2O2), HRP catalyzes the
oxidation of the chromogenic substrate 3,3’,5,5’-tetramethylbenzidene (TMB). This
oxidation of TMB by HRP forms a blue product. Note: TMB is light sensitive, and the
assay results should be determined 5–10 minutes after the substrate is added to the
wells. If the microplate strips sit longer, nonspecific color may develop. Color that
develops after the 5-minute incubation should not be considered in the assay results.
After 20–30 minutes, the blue color may begin to fade as TMB precipitates out of
solution.
Controls: Controls are always run side by side with actual samples to make sure that
the procedure is working correctly. Controls can resolve ambiguous results that occur
due to human error or contaminated reagents; controls must be included in any valid
ELISA. For the negative control, the antigen or primary antibody is either omitted (as
in this kit) or the antigen is replaced by a factor that will not bind specifically to the
antibody. The positive control always contains the target antigen or antibody. A
negative sample that gives a positive assay result is called a false positive. A positive
sample that gives a negative assay result is called a false negative.
Many diagnostic assays give a percentage of false positive or false negative results, so
confirmation of diagnosis by a second type of assay is important. For example,
immunoassays for antibodies to human immunodeficiency virus (HIV) can give either
false positive or false negative results. False positives can result from recent
vaccinations, and false negatives can result from immunosuppression (e.g., from
drugs given after transplants) or from administering the test too soon after infection
with HIV. (Antibodies against HIV do not appear until some weeks after HIV
infection; the appearance of specific antibodies is called seroconversion.)
Because of this, positive HIV ELISA results are always confirmed by western blot
(see page 91). In an ELISA like those in Protocols I and II (in which antigen
concentration is the experimental variable), an appropriate negative control would be
37
wells with antigen omitted. Any color product in those wells would be the result of
either 1) nonspecific binding of the antibodies, or 2) experimental error. An
appropriate positive control would be a sample known to contain the antigen. In an
ELISA antibody test like that in Protocol III (in which primary antibody concentration
is the experimental variable), an appropriate negative control would be wells with
primary antibody omitted. Any color product in those wells would be the result of
either 1) nonspecific binding of the secondary antibody, or 2) experimental error. An
appropriate positive control would be a sample known to contain primary antibody.
For many clinical ELISAs, control solutions are provided with the commercial kits.
Analysis of Results: An ELISA can give qualitative (yes or no) or quantitative (how
much?) information. Qualitative results can be determined visually without the use of
complicated instrumentation. Quantitative results can be estimated visually and scored
symbolically, e.g., (++) for strong signal, (+) for weak signal, (+/–) for an ambiguous
signal, and (–) for no detectable signal. For accurate and precise determination of
concentrations, a microplate reader is required. Microplate readers quantitate the
absorbance of light by the colored substrate in each well of a microplate. They use the
negative control wells to set a baseline and then read the absorbance of each well at a
specified wavelength. For example, the peak absorbance for TMB is at 655 nm.
Quantitative ELISA controls include a dilution series of known concentrations that is
used to create a standard curve. This standard curve allows the concentration of
antigen in a sample to be quantitated, which in turn may help a researcher, clinician,
or physician determine the infection level of a particular disease. A lesson extension
to perform a quantitative ELISA is included in Appendix D.
ELISAs are performed so routinely in both clinical and research laboratories that
assays for many antigens are available in kit form. Kits normally include all
components and controls needed for a given test except for the experimental samples.
For example, Bio-Rad’s Clinical Diagnostics Group produces over 100 kits that are
used to detect autoimmune diseases, blood viruses, genetic disorders,
microorganisms, toxins, and bovine spongiform encephalopathy (BSE or mad cow
disease).
The Bio-Rad ELISA Immuno Explorer kit demonstrates a method to detect the
presence of specific antigens or antibodies in a variety of samples. A number of
different ELISA methods have been developed that differ primarily in the sequence in
which antigens and antibodies are added to the wells. In an antibody capture assay (as
used in this kit), antigen is bound in the plastic wells and the primary antibody binds
to (or is captured by) the immobilized antigen. A secondary antibody is linked to the
enzyme horseradish peroxidase (HRP), which oxidizes its substrate (TMB), turning
the assay solution blue.
38
In an antigen capture assay, primary antibody is bound in the plastic wells, antigen is
captured by the immobilized primary antibody, and the captured antigen is detected
by a secondary antibody, also linked to HRP, that turns the assay solution blue upon
reaction with TMB.
39
Student Manual
Introduction
You are about to perform an experiment in which you will share simulated “body
fluids” with your classmates. After sharing, you will perform an enzyme-linked
immunosorbent assay or ELISA to determine if you have been exposed to a
contagious “disease”. The ELISA uses antibodies to detect the presence of a disease
agent, (for example, viruses, bacteria, or parasites) in your blood or other body fluid.
You will then track the disease back to its source.
When you are exposed to a disease agent, your body mounts an immune response.
Molecules that cause your body to mount an immune response are called antigens,
and may include components of infectious agents like bacteria, viruses, and fungi.
Within days, millions of antibodies — proteins that recognize the antigen and bind
very tightly to it — are circulating in your bloodstream. Like magic bullets, antibodies
seek out and attach themselves to their target antigens, flagging the invaders for
destruction by other cells of the immune system.
Over 100 years ago, biologists found that animals’ immune systems respond to
invasion by “foreign entities”, or antigens. Today, antibodies have become vital
scientific tools, used in biotechnology research and to diagnose and treat disease. The
number of different antibodies circulating in the blood has been estimated to be
between 106 and 1011, so there is usually an antibody ready to deal with any antigen.
In fact, antibodies make up to 15% of your total blood serum protein. Antibodies are
very specific; each antibody recognizes only a single antigen.
How Are Antibodies Made?
Scientists have learned to use the immune response of animals to make antibodies that
can be used as tools to detect and diagnose diseases. The study of the immune system
is called “immunology”. Animals such as chickens, goats, rabbits, and sheep can be
injected with an antigen and, after a period of time, their serum will contain antibodies
that specifically recognize that antigen. If the antigen was a disease agent, the
antibodies can be used to develop diagnostic tests for the disease. In an immunoassay,
the antibodies used to recognize antigens like disease agents are called primary
antibodies; primary antibodies confer specificity to the assay.
Other kinds of antibody tools, called secondary antibodies, are made in the same way.
In an immunoassay, secondary antibodies recognize and bind to the primary
40
antibodies, which are antibodies from another species. Secondary antibodies are
prepared by injecting antibodies made in one species into another species. It turns out
that antibodies from different species are different enough from each other that they
will be recognized as foreign proteins and provoke an immune response. For example,
to make a secondary antibody that will recognize a human primary antibody, human
antibodies can be injected into an animal like a rabbit. After the rabbit mounts an
immune response, the rabbit serum will contain antibodies that recognize and bind to
human antibodies. The secondary antibodies used in this experiment are conjugated to
the enzyme horseradish peroxidase (HRP) which produces a blue color in the
presence of its substrate, TMB. These antibody and enzyme tools are the basis for the
ELISA.
Where Is ELISA Used in the Real World?
With its rapid test results, the ELISA has had a major impact on many aspects of
medicine and agriculture. ELISA is used for such diverse purposes as pregnancy tests,
disease detection in people, animals, and plants, detecting illegal drug use, testing
indoor air quality, and determining if food is labeled accurately. For new and
emerging diseases like severe acute respiratory syndrome (SARS), one of the highest
priorities of the US Centers for Disease Control (CDC) and the World Health
Organization (WHO), has been to develop an ELISA that can quickly and easily
verify whether patients have been exposed to the virus.
Some examples of the use of ELISA in a variety of fields, including veterinary
medicine, food testing, and agriculture are summarized below:
Field
Use
Human and
• Diagnose a variety of diseases, such as West Nile virus (in people or animals),
Veterinary Medicine
HIV, SARS, Lyme disease, trichinosis, tuberculosis, and many more by
detecting serum antibodies
Veterinary
• Detect viruses such as feline leukemia virus (FLV) in cats
• Detect parasites such as heartworms in dogs
• Diagnose thyroid problems in dogs and cats by measuring serum thyroxine
(t4) concentrations
• Diagnose equine encephalitis in horses by detecting Arboviruses
Agriculture: Crops
• Detect viruses such as potato leaf roll virus and cucumber mosaic virus in food
crops
• Detect mycotoxins in crops, such as aflatoxin in cereal grains and corn
• Detect viruses in decorative plants, such as bean yellow mosaic virus in
gladiolus
• Track adulteration of non-genetically modified (non-GMO) crops with GMO
products
Environmental
• Test indoor air quality, such as detecting mold toxins in buildings
Food safety and
• Prevent transmission of bovine spongiform encephalitis (mad cow disease,
quality
BSE) by screening for central nervous system tissues in raw meat, in
processed and cooked meats, and on surfaces
• Determine if food labeling is correct, e.g., by checking for cow milk proteins
in goat milk products or for non-durum wheat in durum wheat products
• Prevent allergic reactions by detecting ingredients that aren't listed on food
content labels, e.g., detecting peanuts in products in which peanuts are not
listed as an ingredient
Other
• Detect restricted or illegal drug use, e.g., performanceenhancing drugs,
marijuana, methamphetamine, cocaine, etc.
• Confirm pregnancy by detecting human chorionic gonadotropin (hCG) in
urine Antigen
41
Some tests give positive or negative results in a matter of
minutes. For example, home pregnancy dipstick tests are
based on very similar principles to ELISA. They detect
levels of human chorionic gonadotropin (hCG), a hormone
that appears in the blood and urine of pregnant women
within days of fertilization. The wick area of the dipstick is
coated with anti-hCG antibody labeled with a pink
compound (step 1). When the strip is dipped in urine, if
hCG is present it will bind to the pink antibody, and the
pink hCG-antibody complex will migrate up the strip via
capillary action (step 2). When the pink complex reaches the
first test zone, a narrow strip containing an unlabeled fixed
anti-hCG antibody, the complex will bind and concentrate
there, making a pink stripe (step 3). The dipsticks have a
built-in control zone containing an unlabeled fixed
secondary antibody that binds unbound pink complex
(present in both positive and negative results) in the second
stripe (step 4). Thus, every valid test will give a second pink
stripe, but only a positive pregnancy test will give two pink
stripes.
Why Do We Need Controls?
Positive and negative controls are critical to any diagnostic test. Control samples are
necessary to be sure your ELISA is working correctly. A positive control is a sample
known to be positive for the disease agent, and a negative control is a sample that
does not contain the disease agent.
Your Task Today
You will be provided the tools and an experimental protocol to perform an ELISA.
You will be given a simulated “body fluid” sample that you will share with your
classmates. One or two of the samples in the class have been “infected”. You will also
be provided with positive and negative control samples. Then you and your fellow
students will assay your samples for the presence of the “disease agent” to track the
spread of the disease through your class population.
Now let’s put this all together.
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The Main Steps in this Antigen Detection ELISA
are:
1. Add your shared sample plus control samples to the
wells in a microplate strip. Your samples contain
many proteins and may or may not contain the
disease agent (antigen). Incubate for 5 minutes to
allow all the proteins in the samples to bind to the
plastic wells via hydrophobic interaction. This is an
immunosorbent assay because proteins adsorb (bind)
to the plastic wells.
2. Add anti-disease antibody (primary antibody) to the
wells and incubate. The primary antibody will seek
out the antigen from the many proteins bound to the
well. If your sample was “infected”, the antibodies
will bind tightly to the disease agent (antigen) in the
wells.
3. Detect the bound antibodies with HRP-labeled
secondary antibody. If the primary antibodies have
bound to the antigen, the secondary antibodies will
bind tightly to the primary antibodies.
4. Add enzyme substrate to the wells, wait 5 minutes,
and evaluate the assay results. If the disease antigen
was present in your sample, the wells will turn blue.
This is a positive diagnosis. If the wells remain
colorless, the disease antigen was not present in your
sample and the diagnosis is negative.
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Pre-Lab Focus Questions
1. How does the immune system protect us from disease?
2.
How do doctors use the immune response to protect you from disease?
3.
What are some ways that diseases spread?
4.
What is an example of a disease that attacks the human immune system?
5.
What problems can prevent the immune system from working properly?
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6.
Why are immunosuppressant drugs necessary when someone has an organ
transplant?
7.
Why is rapid antigen detection test necessary?
8.
What does ELISA stand for?
9.
Why are enzymes used in this immunoassay?
10. Why do you need to assay positive and negative control samples as well as your
experimental samples?
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Laboratory Guide
Student Workstation Checklist
One workstation serves 4 students
Items
Contents
Yellow tubes
Student samples (0.75 ml)
Violet tube (+)
Blue tube (-)
Green tube (PA)
Orange tube (SA)
Brown tube (SUB)
12-well microplate strips
50 μl fixed-volume micropipette or
20–200 μl adjustable micropipet
Positive control (0.5 ml)
Negative control (0.5 ml)
Primary antibody (1.5 ml)
Secondary antibody (1.5 ml)
Enzyme substrate (1.5 ml)
Disposable plastic transfer pipet
70-80 ml wash buffer in beaker
Phosphate buffered saline
with 0.05% Tween 20
Large stack of paper towels
Black marking pen
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Number
4 (1 per
student)
1
1
1
1
1
2
()
10-20
☐
5
☐
1
☐
2
1
☐
☐
☐
☐
☐
☐
☐
☐
☐
Laboratory Procedure
1. Label the yellow tubes with each student’s initials
2. Label the outside wall of each well of your 12-well strip. Two students may share a
strip of 12 wells. On each strip, label the first three wells with a “+” for the positive
controls and the next three wells with a “–” for the negative controls. On the
remaining wells write your and your partner’s initials. For example, Florence
Nightingale and Alexander Fleming would label their shared strip like this:
3. Bind the antigen to the wells:
a. Use a pipet to transfer 50 μl of the positive control (+) from the violet tube into
the three “+” wells.
b. Use a fresh pipet tip to transfer 50 μl of the negative control (–) from the blue
tube into the three “–” wells.
c. Use a fresh pipet tip for each sample and transfer 50 μl of each of your team’s
samples into the appropriately initialed three wells.
4. Wait 5 minutes while
the samples bind to
all the proteins in
the plastic wells.
5. Wash the unbound sample out of the wells:
a. Tip the microplate strip upside down onto the paper towels so that the samples
drain out, then gently tap the strip a few times upside down on the paper
towels. Make sure to avoid samples splashing back into wells.
b. Discard the top paper towel
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c. Use a fresh transfer pipet filled with wash buffer from the beaker to fill each
well with wash buffer taking care not to spill over into neighboring wells. The
same transfer pipet will be used for all washing steps.
d. Tip the microplate strip upside down onto the paper towels so that the wash
buffer drains out, then gently tap the strip a few times upside down on the
paper towels.
6. Repeat wash step 5.
7. Use a fresh pipet tip to transfer 50 μl of primary antibody (PA) from the green tube
into all 12 wells of the microplate strip.
8. Wait 5 minutes for the primary antibody to bind.
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9. Wash the unbound primary antibody out of the wells by repeating wash step 8 two
times.
10. Use a fresh pipet tip to transfer 50 μl of secondary antibody (SA) from the orange
tube into all 12 wells of the microplate strip.
11. Wait 5 minutes for secondary antibody to bind.
12. Wash the unbound secondary antibody out of the wells by repeating wash step 4
three times.
The secondary antibody is attached to an enzyme (HRP) that chemically changes the
enzyme substrate, turning it from a colorless solution to a blue solution. Predict which
wells of your experiment should turn blue and which should remain colorless and
which wells you are not sure about.
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13. Use a fresh pipet tip to transfer 50 μl of enzyme substrate (SUB) from the brown
tube into all 12 wells of the microplate strip.
14. Wait 5 minutes. Observe and record your results.
Results Section
Label the figure below with the same labels you wrote on the wells in step 1. In each
of the wells, put a “+” if the well turned blue and a “-“ if there is no colour change.
Is your sample positive, explain your answer.
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Post-Lab Focus Questions
1. Did your sample contain the antigen
2. The samples that you added to the microplate strip contain many proteins and may
or may not contain the antigen. What happened to the proteins in the plastic well if
the sample contained the antigen? What if it did not contain the antigen?
3. Why did you need to wash the wells after every step?
4. When you added primary antibody to the wells, what happened if your sample
contained the antigen? What if it did not contain the antigen?
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5. When you added secondary antibody to the wells, what happened if your sample
contained the antigen? What if it did not contain the antigen?
6. If the sample gave a negative result for the antigen, does this mean that the antigen
is not present? What reasons could there be for a negative result when the antigen
is actually present?
7. Why did you assay your samples in triplicate?
8. What antibody-based tests can you buy at your local pharmacy?
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9. Antigen Detection ELISA for detecting the presence of FeLV p27 core protein
in peripheral blood (Summary)
Briefly describe the main steps (1, 2, 3 & 4) of an antigen detection ELISA for
FeLV as illustrated in the figure below.
NOTE: The wash steps are omitted
1.
2.
3.
4.
10. Name the FeLV antigen that the antigen detection ELISA for FeLV is detecting
in peripheral blood in FeLV-infected cats.
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Exercise 7. Bioinformatics.
Introduction
Bioinformatics is the analysis and use of sequence data from nucleic acids and
proteins. There is not much point in knowing the DNA sequence of a gene or
organism if it is a just a string of A, C, G and T’s. Similarly, if we know what the
amino acid sequence is in the primary structure of a protein what does it mean and
can it tell us anything? What we do with that sequence data can tell us a lot about
the gene or organism from which it came and its evolutionary relationship to other
similar genes or organisms. Along with discovering methods to sequence DNA
and proteins was the development of software for storage and analysis of the data.
Considerable computing power is necessary for comparison of entire genome
sequences of higher organisms however comparing sequences of genes with
others takes much less and can be done with relatively simple PC based
programs.
Much of the sequence data produced to data is in the public domain and anyone
can access it if they have a computer and access to the internet. Two major
databases freely available are maintained by the US National Centre for
Biotechnology Information (NCBI), and the European Molecular Biology
Laboratory (EMBL). NCBI maintains the Genbank database containing all DNA
sequences published or submitted to date. Large protein sequence databases like
SwissProt are linked to from the NCBI. The website for the NCBI is
http://www.ncbi.nlm.nih.gov/ and it worth a look around as there are many other
resources such as a searchable literature database with access to many free
journals (PUBMED).
The NCBI in addition to hosting these databases provide online programs so that
researchers can put in their favourite sequence data and analyse them. This
exercise will introduce you to some of these programs and databases. There are
two exercises is in the resources of the interact site along with the sequences you
will require for completing these exercises. You can do these exercises yourself at
home, but we will also go through them and discuss results in the laboratory
period. Please record the information you find out and answer the questions from
the exercise below. You will be examined on this in the end of session exam.
Answer to questions
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