Components of Lysis Buffer
• Buffer (Tris or Hepes buffer with pH 7-8)
• Salt (usually NaCl 150mM (low) to
500mM(high)
• Chelating agent (EDTA or EGTA)
• Detergent
• Protease inhibitor
• Phosphatase inhibitor (optional)
Lysing Cells
• Treat cells with appropriate conditions depending
on the experiment
• Pellet cells and lyse them in the appropriate lysis
buffer.
• Most important ingredient in lysis buffer is
detergent.
– Most stringent to weakest
•
•
•
•
•
•
•
SDS
NP-40
Triton X-100
Tween 20
Digitonin
CHAPS
Brig
• Second most important ingredient is protease
inhibitors.
– Once proteins are denatured by detergents, they are
susceptible to degradation by proteases.
– Need more than one inhibitor since there are lots of
proteases.
– Protease inhibitors
• Aprotinin
• Leupeptin
• PMSF (phenylmethylsulfonyl fluoride)
– Add immediately before lysis since PMSF activity decreases over
time in aqueous solutions. About 15-30 minutes of activity.
Phosphatase inhibitors
• Need to add to lysis buffer if using
phopshospecific antibodies or suspect protein of
interest is phosphorylated
• Inhibitors
– ZnCl2
– NaF
– Na-Vanadate (tyrosine phosphatase inhibitor, add prior
to lysis since only active in pH 7 for minutes)
Further denature proteins
• Add lysate either a known concentration of proteins
or cell number equivalent to SDS loading buffer
• SDS Loading Buffer
–
–
–
–

Buffer (Tris-Cl pH 6.0)
2% SDS
0.1% bromophenol blue
10% glycerol (allows sol’n to sink to bottom of gel wells)
b-mercaptoethanol ( reducing agent)
• SDS loading gel mixed with lysate is boiled to further
denature proteins.
• 1:10 ratio loading buffer to lysate
Western blotting
• Detects proteins and estimates their
molecular weight.
• Detects changes in phosphorylation and
lipid modifications.
• Used to detect changes in protein
expression.
SDS-Polyacrylamide Gel
Electrophoresis of proteins
• This gel ensures that proteins are
dissociated into their polypeptide subunits
and minimizes aggregation.
• Proteins migrate on gel according to their
molecular weight therefore if proteins of
know amounts are added to the gel the
approximate molecular weight of your
protein of interest can be found.
SDS-Polyacrylamide Gel
Electrophoresis of proteins
• Protein lysate is now loaded onto a SDSpolyacrylamide gel electrophoresis apparatus.
• This apparatus contains a gel that separates protein
out according to their molecular weight.
• GEL
–
–
–
–
15%
10%
7.5%
5.0%
12-43kDa
16-68kDa
36-94kDa
57-212kDa
Types of electrophoresis
apparatus
• Large
– gels can be made with 10 or 15 wells
– Hold up to 200ml of sample
– Runs for 4 hours or overnight
• Small
– Gels can be made with 10 or 15 wells
– Hold up to 50ml of sample
– Runs for 2 hours
Ingredients in Gel
• Sodium dodecyl sulfate (SDS)
• Tris buffer (either glycine or tricine)
• Acrylamide and NN-bis-acrylamide
– Forms gel matrix
• TEMED
– Catalyst for polymerization (produces free radials from
APS)
• Ammonium persulfate (APS)
– Source of free radials for polymerization
Could purchase pre-cast gels if you have the
money.
Pouring your gel
• Pour running gel first. Contains Tris buffer
at pH 8.0. The percent of acrylamide may
be adjusted for better resolution of proteins
(5-15%)
– Pour ethanol or distilled water on top of gel for
even polymerization.
– Leave enough room to pour stacking gel, about
one forth of total gel.
Pouring gel continues
• After the running gel has polymerized, the gel is
washed with distilled water to remove any debris
on the gel to give a good interface between
stacking and running gels. The stacking gel is
pour. It contains Tris buffer at pH 6.8.
– 5% acrylamide for maximum porosity
– Deposits proteins on stacking and running gel interface
which concentrates the proteins and provides better
resolution.
– Insert comb to form wells at an angle to prevent air
bubbles.
Loading gel
• Remove comb and wash wells out with running
buffer.
• Best to use loading tips (Hamilton syringes also
work) to load samples.
• Start at the bottom of well and work your way up
the well.
• Glycerol in the loading buffer will keep sample in
well.
• Optional- Running buffer left in wells or wells
empty.
Running Gel
• After loading samples, added running buffer to
upper reservoir and lower reservoir. Hint. Add
upper reservoir first to detect leaks.
• Running buffer provides the ions to conduction
the current through the gel.
• SDS makes proteins negatively charged that
attaches the proteins to the anode.
• Therefore in electrophoresis, the current must run
from cathode (negatively charged, black) to the
anode (positive charged, red).
After the gel has run
• Remove gel from the glass plate holders
• Place the gel on filter paper and added
nitrocellulose on top of the gel.
• Optional– submerged in transfer buffer
– Soaked in transfer buffer
• Transfer buffer contains Tris, glycine and
methanol but no ions. Ions will coat the electrodes
and destroy the transfer apparatus.
• Gel:nitrocellulose must have no air bubbles
between them (proteins will escape into the bubble
not into the nitrocellulose.)
Transfer apparatus
• Two major types
– Semi-dry apparatus
• No transfer buffer chamber
• Only has plates (runs for hours)
– Submerge transfer apparatus
• Contains a chamber filled with transfer buffer
• Electrodes
– Plate electrodes (runs for hours)
– Wire electrodes (runs overnight)
Running a transfer
• Similar to running a gel
– Negatively charged proteins run towards the anode end
of the transfer apparatus
– Always have nitrocellulose on the anode side to capture
proteins.
• Is nitrocellulose your only choice?
– PDFV is another membrane
• Why use it?
– Stronger than nitrocellulose and able to strip phosphate groups
off proteins.
– Must soak in methanol first since it doesn’t become wet in water
After transfer
• Membrane is washed in a Tris, borate, saline
solution.
– Options
• Added detergent Tween 20 to reduced background in western
blotting.
• Block membrane in non-fat dried milk solution.
Usually 5% w/v. Prevents binding of antibodies to
membrane.
– Don’t buy milk from Fisher
– Safeway brand or Carnation brand are the best
– Alternatively, use bovine serum albium (BSA) but its
more expensive (not available in Supermarkets)
Antibodies
• Proteins that bind to specific epitopes on specific
proteins.
• Two main types used in western blotting
– Monoclonal
– Polyclonal
• Monoclonal are generally produced from mice and
polyclonal are produced from rabbit or goat but
other animals also produced polyclonals.
What antibodies are best for
western blotting?
• Both types work for western blotting but
generally polyclonals are better
– Monoclonals are to one epitope while
polyclonals are to many epitopes on the protein
of interest. If epitope changes upon denaturing
of the protein it will not recognize the protein
– Each antibody has its own characteristics so it
is hard to predict what antibodies work well in
a western and which are bad.
Primary Antibodies
• After blocking membrane, add antibodies in
concentrations recommended by manufacture or in
dilution series if unknown to blocking solution (ie
5% milk).
• Most antibodies are added as a dilution such as
1:1000.
• Incubate overnight at 4oC or 2 hours at room
temperature (only if it’s a good antibody).
• Wash in TBS with detergent Tween 20 (TBST)
Secondary Antibodies
• Wash at least three times with TBST for 5-10
minutes each
• Added antibodies against the animal that the
primary antibodies were made in.
• These antibodies are also conjugated with
enzymes such as horse radish peroxidase
• The antibodies are added to blocking buffer and
incubated at room temperature for one hour.
Detection of your protein
• After secondary antibody incubation wash
with TBST as before.
• Add enhance chemiluminance reagents
(commercially available) that have
substrates for HRP that gives a product that
gives off light energy.
• Place on autorad film and bands appear.
HEK 293
Normal
WESTEN BLOT FOR MCL-1 PROTEIN AND
REPROBED FOR ACTIN
1
2
3
4
5
6
Patients
Mcl-1
b-actin
SNAP i.d.® Protein Detection System
• System provides a vacuum to increase the
interaction of antibody to protein in the
membrane.
• Reduces incubation times down from 1 hour to 10
minutes.
• Potentially reduces background
• Uses same blocking and antibodies as standard
method.
• Antibody concentration increased to at least 1:300
from standard 1:1000.
SNAP ID Vaccuum Apparatus
Analysis of results
• Many antibodies bind to nonspecific bands.
• Use recombinant proteins to verify specificity of
your antibody.
• If changes occur upon treatment in the molecular
weight of your protein, its an indication of
specificity of the antibody.
• Use another antibody to a protein to confirm equal
loading of protein. Alternatively, stain membrane
with Ponceau S that detects proteins.
Analysis continued
• If protein band is fussy or diffuse, it might
indicate lipid modification.
• If protein has multiple bands, it might
indicate phosphorylation or alternate spliced
isoforms.
What could go wrong?
• Retrophoresis
• Eastern blot
• Western is blank
– Decrease blocking solution concentration
– Increase protein loaded on gel
– Change antibodies
• Western background very high
– Solution is to increase Tween 20 in TBST and/or use another blocking
solution.
– Increase secondary antibody dilution
– Increase protein concentration loaded on gel
– Use another antibody
– Use enhance chemifluorescence (ECF) instead of ECL
Is the membrane usable after
western blotting?
• Yes
– Can reprobe membrane with another antibody
• Strip the membrane of any attached antibodies from the first
western (striping buffer contains SDS, and b-mercaptoethanol)
• Wash membrane thoroughly to remove any striping buffer
• Reblock the membrane and repeat the western with a new
primary antibody.
• Remember that 20% of proteins on the membrane are lost by
striping the blot so only can do this once or twice.
Experimental Design Hints
• Always have a untreated or normal protein
control.
• Always check for equal protein loading
• Load either equal amount of protein or
equal cell equivalence.
• Moneysaving tip
– Can reuse the primary antibody in blocking
solution up to three times.
Experimental design (continued)
• If specificity of antibody is in question, add
recombinant protein to the antibody solution
and then western blot with this antibody.
The protein of interest will not show in the
western blot using antibody mixed with
recombinant protein compared to using the
antibody alone solution.
• Run recombinant protein on gel.
Immunoprecipitation
• This technique allows proteins undetectable
by western blotting to be visualized.
• Analysis of associating proteins.
• Analysis of post-translational modifications.
Preparing beads
• Obtain commercially available beads
containing protein A or protein G
conjugated to them.
• Protein A and Protein G binds
immunoglobulins of specific subclass and
species in the Fc region.
Pre-clear beads
• Beads usually are obtained in a slurry
solution. Remove beads ( usually 25-50ml)
for the stock.
• Wash beads with rabbit or mouse serum
prior to immunization if antibody being
used is polyclonal.
Pre-clear continued
• Beads can also be incubated with heatkilled formaldehyde-fixed staphylococcal
aureus.
• Wash beads at least three times with lysis
buffer used to lyse cells with.
Antibody:bead complexes
•
•
•
•
Resuspend beads in lysis buffer
Added the appropriate primary antibody.
Incubate by rotation at 4oC for one hour.
Alternatively, add antibody to lysate and
incubate by rotation at 4oC for one hour.
Beads:Antibody:Protein
Complex
• After one hour incubation, the
bead:antibody complex is washed three
times with lysis buffer.
• Lysate is added to the bead:antibody
complex and incubated one hour at 4oC.
• After one hour beads:antibody:protein
complex is washed again with lysis buffer.
Immunoprecipitation
Caution: May lose beads in
washing procedure
• Make sure you leave a little buffer in the
tube.
• Alternative method:
– Using a magnetic bead that will stick to side of
tube that contains a magnet.
– Remove magnet and beads can be resuspended.
Isolate protein
• The bead complex contains a small volume of
lysis buffer (50ml)
• SDS loading buffer is added and the beads are
boiled for 10 minutes
• The proteins are stripped off the complex by
strong detergent (SDS), reducing agent (bmercaptoethanol) and heat (100oC).
• Protein ready to load on gel for western blotting.
Question: What if your beads don’t
recognize your primary antibody
• Answer: Add antibodies against the species
of the primary antibody that recognize
protein A or G (ie rabbit anti mouse against
a mouse monoclonal IgG1isotype)
Applications
• Visualize proteins not detectable by western
blotting since immunoprecipitation
concentrates the lysate. For example, 1mg
of protein can be added to bead complexes
while only 0.5mg can be loaded on to a gel
for western blotting.
Application (continued)
• Protein-Protein interactions
– When protein is isolate associating proteins are
also isolated and concentrated. The sample is
then western blotted for the associating protein.
– If associating protein is not known, proteins can
be labeled with radioactive amino acids and
detected on a SDS-polyacrylamide gel by X-ray
film.
Co-immunoprecipitation
Application (continued)
• Western blotted for post-translational
modifications such as tyrosine
phosphorylation. Reduces or eliminates
other proteins at the same molecular weight
that might also be post-translationally
modified.
• Analysis protein for enzymatic activity
(Kinase Assay).
Controls
•
•
•
•
•
Only bead:antibody complexes
Only bead:lysate complexes
Beads alone
Primary antibody against unrelated protein
If western blotting for associating proteins
or post-translational modifications, must
strip blot and reprobe with antibodies
against immunoprecipitated protein.
Analysis of immunoprecipitation
• Antibodies used in immunoprecipitation
will also be separated on the gel and
recognized by secondary antibody.
– Solution is to use TrueBlotTM that reduces
immunoglobulin bands (expensive).
• Eliminate non specific bands with controls
• Use lysate or recombinant protein to
confirm molecular weight of protein.
Immunoprecipitation and western with ptyr
antibodies
Tyrosine kinase Assay
Problems
• Not all antibodies are able to
immunoprecipitate proteins
• Background is high
• Large number of non-specific bands.
• Low expression of protein
• No tyrosine phosphorylation detected.
Solutions
• Use different antibodies
• Change detergent in lysis buffer
• Change protein concentration loaded on
beads.
• Change antibody concentration in
immunoprecipitation.
• Change blocking buffer from milk to BSA.
Question: What happens if your protein
of interest is the same or close to the
same molecular weight as the
immunoprecipitating antibody
• Answer: Add loading buffer without reducing
agent. The antibody complex will be at the top of
the gel and the protein will be detectable.
• Alternative: Use TrueBlot or similar products to
reduce immunoglobulin bands without blocking
protein of interest binding.
TRUEBLOT METHOD
TrueBlot: Free your IP/WB of denatured light & heavy chains!
Figure 1: Stat1 was immunoprecipitated from 0.5 ml of 1x10 7 Jurkat cells/ml with 3 µg
rabbit anti-human Stat1. Precipitate from 5x105 cells was subjected to
electrophoresis, transferred to an Immobilon membrane, and immunoblotted with
anti-Stat1 using Rabbit TrueBlot™ and conventional HRP-conjugated detection.
Lane 1: Detection with Rabbit IgG TrueBlot™ - note the absence of the anti-Stat1
immunoprecipitating heavy and light chains. Lane 2: re-blot of lane 1 using the
conventional HRP-conjugated detection anti-rabbit polyclonal antibody - note the
presence of the Stat1 immunoprecipitating heavy and light chains confirming that
although the immunoprecipitating heavy and light chains were present in the
sample in lane 1, Rabbit IgG TrueBlot™ detected native antibody but not the
denatured heavy and light chains.
What happens if
immunoprecipitation antibody does
not exit?
• Recombinant protein with a tag (GST, HA, His or Myc)
• Purify from bacteria, mammalian cells or in vitro
translational assay (produce protein in a tube).
• Bind to bead with antibodies against tag or a nickel column
for His tag.
• Run lysate from target cells over beads (associating protein
will bind to recombinant protein). Can be done both in vivo
or in vitro.
• Run target binding protein (recombinant) over the bead
complexes.
What happens if immunoprecipitation
antibody does not exit?
• Western blot for targeted proteins if known.
• If unknown, release complexes from beads
and run sample on a mass spectrometer.
– Note: This will also work for traditional
antibody mediated immunoprecipitations.
• Always run at tag and bead alone control.
Problems:
• Recombinant protein are susceptible to
breakdown (naïve versus denatured).
• Bead complexes could be binding proteins
non-specifically.
• Bacterial or mammalian cells could induce
protein-protein complexes that are not
physiologically relevant due to over
expression.
Can you look at DNA-protein
interactions?
• YES, crosslinking proteins to DNA will
provide the complexes for
immunoprecipitation.
• This technique is called chromatin
immunoprecipitation (ChIP).
Chromatin Immunoprecipitation
(ChIPs)
DNA-binding proteins are crosslinked
to DNA with formaldehyde in vivo.
Shear DNA along with bound proteins
into small fragments.
Bind antibodies specific to the DNAbinding protein (eg. Histones)
Isolate the complex by precipitation
Reverse the cross-linking
Release the DNA & digest the proteins
PCR amplify the specific DNA
sequences of the desired gene (DR5).
NFkB binds to DR5 gene following
both etoposide and EGF
stimulation (ChIP assay)
Etoposide
C
2h
4h
EGF
8h
C
1h
2h
4h
DR5
Control
Input
i.p. p65
Problems
• Only a fraction of protein bind DNA so
immunoprecipitation must be efficient.
• Crosslinking could cross-link protein-proteinprotein-DNA artificial complexes.
• Time dependent for protein binding to DNA.
• PCR product must be small and specific for target
DNA. Non-specific PCR could give false
positives.
Can you look at RNA-protein
interactions?
• YES, crosslinking proteins to RNA will
provide the complexes for
immunoprecipitation.
• This technique is called crosslinking and
immunoprecipitation of RNA-protein
complexes (CLIP).
• Same limitations with the added problem of
RNA degradation.
Group Question
• Protein X expression (65kDa) is increased
following activation of the T cell receptor.
• Antibody against Protein X is rabbit polyclonal
• Design an experiment where you can detect the
increase in protein X levels
–
–
–
–
–
What detergent in lysis buffer to use?
Western versus Immunoprecipitation?
What percent gel to use?
What secondary antibody to use?
Any controls?