Protocol/Immunoprecipitation

Protocol for Immunoprecipitation

Materials and Reagents

Lysis buffer

• 25 mM Tris-HCl, pH 7.5
• 150 mM NaCl
• 1 mM Sodium orthovanadate
• 20 mM NaF
• 5 mM EDTA
• 1 mM EGTA
• 2% Triton X-100
• 10% Glycerol
• 1 mM ZnCl2

Stored and stable at 4°C. Before use,

Add

• 1 mM Phenylmethylsulfonyl fluoride (PMSF) PMSF is very labile in water. A more expensive but more stable alternative is 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) (Pefabloc; Roche Molecular Biochemicals, Mannheim, Germany).
• 10 μg/mL Pepstatin
• 10 μg/mL Leupeptin
• 10 μg/mL Aprotinin

Washing Buffer

• 50 mM Tris-HCl, pH 7.5
• 0.3 M NaCl
• 0.5% Triton X-100
• 0.02% NaN3

The washing buffer can be stored at 25°C for a number of months.

• 0.1 M Sodium orthovanadate (phosphatase inhibitor). Dissolve powder in water at pH 10.0. Boil. Keep at room temperature for up to 1 week in the dark.
• 10 mg/mL of aprotinin and leupeptin in water. Keep frozen at -20°C in aliquots.
• 10 mg/mL of pepstatin A in ethanol. Keep frozen at -20°C in aliquots.
• Phosphate-buffered saline (PBS) 50% Protein A-Sepharose Solution Resuspend protein A-sepharose slurry in PBS (100 mg of protein A-sepharose powder once hydrated corresponds to 400 μL of volume). Wait until all crystals are dissolved. Pellet by centrifugation (14 000× g) for 10 minutes at full speed in a centrifuge at 4°C. Discard supernatant and resuspend in PBS containing 1% Triton X-100. Repeat centrifugation. Discard the supernatant and add to the pellet an equal volume of immunoprecipitation buffer containing 100 μg/mL BSA and 0.1% sodium

2× Electrophoresis Buffer

• 250 mM Tris, pH 6.8
• 100 mM Glycine
• 4% Sodium dodecyl sulfate (SDS)
• 10% Glycerol

Procedure

The procedure here assumes that a concentration step is required to obtain enough protein material for the immunoprecipitation analysis. It is possible to lyse 2 to 4 100-mm diameter dishes using 1 mL of lysis buffer in order to concentrate the protein content.

1. Decant the medium and follow with a rapid rinse in PBS. After the cells are washed, drain and aspirate the excess PBS.

2. Add 250 to 350 μL of lysis buffer to the plate. Scrape the cells from the dish and transfer them to a microcentrifuge tube. The viscosity of the sample can be reduced by a brief sonication or by several passages through a 26 gauge needle.

3. Leave the sample on ice for 30 minutes.

4. Centrifuge (14 000× g) at 4°C for 10 minutes at 14 k rpm in a microcentrifuge. 5. Collect the supernatant and measure the protein concentration using a BCA kit and protein standards (Pierce Chemical, Rockford, IL, USA). The cell lysates can be stored at -80°C. In many protocols, a preclearing step is performed to remove molecules that bind nonspecifically to the insoluble protein A or protein G (steps 7–8).

6. Use 1 to 2 mg (in a 1000 μL volume) of total proteins for immunoprecipitation.

7. Add 25 μL of protein-A-sepharose solution (shake to suspend slurry before pipetting) and incubate on a tube turner for 1 hour at room temperature.

8. Centrifuge (14 000× g) for 1 minute at 14 k rpm in a microfuge and transfer the supernatant to another tube.

9. Add 1 to 5 μg of antibody to each tube and incubate for 4 hours at room temperature or overnight at 4°C.

10. Add 100 μL of protein A-sepharose solution (shake to suspend slurry before pipetting) and incubate on a tube turner for 3 hours at room temperature.

11. Centrifuge (14 000× g) for 1 minute at 14 k rpm in a microcentrifuge and retain the pellet.

12. Add 500 μL of cold washing buffer, vortex mix, spin for 1 minute at 14 k rpm in a microcentrifuge, and discard the supernatant.

13. Add 1 mL washing buffer, vortex mix, spin for 1 minute, discard the supernatant, and repeat 2 times.

14. Add 1 mL 10 mM Tris-acetate, pH 7.5, vortex mix, spin for 1 minute, and discard the supernatant.

15. Solubilize all samples in 30 to 50 μL of the SDS sample buffer, vortex mix, boil for 5 minutes, and centrifuge.

16. Save the supernatant. Immunoprecipitates in sample buffer can be stored almost indefinitely at -80°C. Storage for more than 7 to 10 days at -20°C can lead to deterioration.

17. Electrophorese the sample on a SDSpolyacrylamide gel and transfer the proteins to a polyvinylidene fluoride (PVDF) membrane (Cat. No. 1722026; Roche Molecular Biochemicals). To prevent tyrosine dephosphorylation during the transfer procedure, we recommend adding 100 μM sodium orthovanadate to the transfer buffer.

18. Incubate the membrane in 50 mL of blocking buffer for 1 hour at room temperature. We recommend not using a blocking buffer that contains dry milk because antiphosphotyrosine antibodies bind to a number of the milk proteins. The following blocking buffer can be used: 5% (wt/vol) BSA, 10 mM Tris-HCl, pH 7.4, 0.15 M NaCl.

19. Incubate the membrane in antiphosphotyrosine antibodies in blocking buffer for 2 hours at room temperature or overnight at 4°C. Several antiphosphotyrosine antibodies are commercially available. We recommend monoclonal antibodies (PY20; Transduction Laboratories, Lexington, KY, USA; or 4G10; Upstate Biotechnology, Lake Placid, NY, USA). It is possible to make a mixture of the two antibodies (PY20 1:1000 dilution, 4G10 1:2500 dilution).

20. Wash the membrane for 1 hour with TBST (Tris-buffered saline with Tween) at room temperature with agitation. Replace the washing solution every 10 to 15 minutes.

21. Incubate the membrane with horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature.

22. Wash the membrane as described in step 20.

23. Detect by using ECLplus reagent (Amersham Pharmacia Biotech) following the manufacturer’s instructions. azide. Store at 4°C for up to 6 months. 2× Electrophoresis Buffer

• 250 mM Tris, pH 6.8
• 100 mM Glycine
• 4% Sodium dodecyl sulfate (SDS)
• 10% Glycerol

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