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) -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 SDS-polyacrylamide gel electrophoresis apparatus.

• This apparatus contains a gel that separates protein out according to their molecular weight.

• GEL– 15% 12-43kDa– 10% 16-68kDa– 7.5% 36-94kDa– 5.0% 57-212kDa

Types of electrophoresis apparatus

• Large – gels can be made with 10 or 15 wells– Hold up to 200l of sample– Runs for 4 hours or overnight

• Small– Gels can be made with 10 or 15 wells– Hold up to 50l 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.

Nor

mal

HE

K 2

93

1 2 3 4 5 6 Patients

Mcl-1

-actin

WESTEN BLOT FOR MCL-1 PROTEIN AND REPROBED FOR 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 -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 25-50l) 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 heat-killed 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 (50l)

• 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 (-mercaptoethanol) 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: Free your IP/WB of denatured light & heavy chains!

Figure 1: Stat1 was immunoprecipitated from 0.5 ml of 1x107 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.

          

                                  

TRUEBLOT METHOD

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.

• 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.

What happens if immunoprecipitation antibody does not exit?

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 DNA- binding 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)

C 2h 4h 8h

i.p. p65

EGF

C 1h 2h 4h

DR5

Control

Input

EtoposideEtoposide

Problems

• Only a fraction of protein bind DNA so immunoprecipitation must be efficient.

• Crosslinking could cross-link protein-protein-protein-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?