THANK YOU FOR THE CHANGE IN TIME

1. THANK YOU FOR THE CHANGE IN TIME

2. Learning Objectives • To learn the methods and procedures involved in western blotting and immunprecipitation. • Understand the problems and controls to design a proper experiment using western blotting and/or immunoprecipitation.

3. 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)

4. 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

5. 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.

6. 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)

7. 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

8. Western blotting • Detects proteins and estimates their molecular weight. • Detects changes in phosphorylation and lipid modifications. • Used to detect changes in protein expression.

9. 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.

10. 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

11. 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

12. 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.

13. 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.

14. 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.

16. 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.

17. 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).

18. 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.)

19. 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)

20. 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

21. 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)

22. 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.

23. 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.

24. 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)

25. 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.

26. 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.

27. WESTEN BLOT FOR MCL-1 PROTEIN AND REPROBED FOR ACTIN HEK 293 Normal 1 2 3 4 5 6 Patients Mcl-1 b-actin

28. 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.

29. SNAP ID Vaccuum Apparatus

30. 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.

31. 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.

32. 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

33. 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.

34. 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.

35. 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.

36. Immunoprecipitation • This technique allows proteins undetectable by western blotting to be visualized. • Analysis of associating proteins. • Analysis of post-translational modifications.

37. 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.

38. 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.

39. 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.

40. 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.

41. 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.

42. Immunoprecipitation

43. 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.

44. 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 (b-mercaptoethanol) and heat (100oC). • Protein ready to load on gel for western blotting.

45. 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)

46. 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.

47. 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.

48. Co-immunoprecipitation

49. 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).

50. 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.