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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PROTEIN DETECTION: GEL ELECTROPHORESIS

IMMUNOPRECIPITATION OF PROTEINS FROM RADIOLABELED CULTURES

Immunoprecipitation of Proteins from Radiolabeled Cultures Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

Overview Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

There are several different lysis buffers that are presented in this protocol. It may require some testing of the different lysis buffers to determine those buffers that extract the protein of interest most efficiently.

Procedure Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

1. Radiolabel culture (see Protocol ID#913 and Protocol ID#505; see Hint #2 and Hint #3). 2. Remove the radiolabeled culture from the incubator and aspirate the media into a radioactive waste bottle (CAUTION! See Hint #3). 3. Chill the cells on ice and rinse twice with TBS (for S35) or PBS (for P32). 4. Add Lysis Buffer:    0.4 ml for a 16 mm well    0.8 ml for a 35 mm well    1.5 ml for a 100 mm plate 5. Scrape the cells off the tissue culture plate with a teflon scraper and transfer to a large polycarbonate centrifuge tube. 6. Add Lysis Buffer to the culture dish:    0.1 ml for a 16 mm well    0.2 ml for a 35 mm well    0.5 ml for a 100 mm plate 7. Rescrape the dish and pool the samples with the previous step. 8. Centrifuge the samples for 10 min at 10,800 X g at 0 to 4°C (see Hint #4). 9. Carefully remove the supernatant, keeping the tube at an angle so as not to disturb the pellet. 10. Samples may be frozen at -70°C at this point (see Hint #5). 11. Counts are usually performed at this time. Use 2 μl for TCA filter precipitations (see Protocol ID#1945). The acid precipitable counts and lysis buffer volumes are usually equalized for analytical comparisons. 12. After the samples are equalized, the antibody is added to the sample (see Hint #6). 13. Incubate at 0°C for 45 minutes to 1 hr with occasional mixing. 14. Add 20 volumes of 10% Staph A per volume of antibody. 15. Incubate for 15 minutes at 0°C with occasional mixing. 16. Centrifuge in a microcentrifuge at maximum speed for 2 minutes at 0 to 4°C. 17. Without disturbing the pellet, carefully discard the supernatant into a radioactive waste container. 18. Wash the pellet by adding 0.5 ml of the Immunoprecipitation High Salt Wash Buffer. Resuspend the pellet in the buffer with a pipette tip and centrifuge in a microcentrifuge at maximum speedfor 2 min at 0 to 4°C. 19. Repeat the wash step with the Immunoprecipitation Low Salt Wash Buffer. 20. Repeat the wash step with the Immunoprecipitation No Salt Wash Buffer. 21. Resuspend the pellet in 60 to 75 μl of SDS-Sample Buffer. 22. Incubate at room temperature for 20 min. 23. Centrifuge the samples for 8 min in a microcentrifuge at maximum speed. 24. Collect the supernatant. 25. Boil the supernatant for 2 to 3 minutes. Centrifuge for 0.5 to 1 min in a microcentrifuge at maximum speed. 26. Electrophorese the samples on a SDS polyacrylamide gel (see Protocol ID#455). 27. Analyze results by autoradiography (see Protocol ID#9036).

Solutions Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

SDS-Sample Buffer    62.5 mM Tris, pH 6.8 5% (v/v) 2-Mercaptoethanol 10% (v/v) Glycerol 2.3% (w/v) SDS 0.001% (w/v) Bromophenol Blue Staph A    10% (v/v) Staph A in the appropriate lysis buffer Modified Opperman Lysis Buffer    1% (v/v) NP40 100 mM KCl 10 mM Potassium Phosphate, pH 7.4 1 mg/ml BSA 1% (w/v) Aprotinin 1 mM EDTA Immunoprecipitation No Salt Wash Buffer    10 mM Tris, pH7.5 0.1% (v/v) NP40 Opperman Lysis Buffer    1% (v/v) NP40 10 mM Tris, pH 7.4 100 mM NaCl 1% (w/v) Aprotinin 1 mM EDTA 1 mg/ml Bovine Serum Albumin (BSA) Immunoprecipitation Low Salt Wash Buffer    1% (v/v) NP40 100 mM NaCl 1 mM EDTA (See Hint #1) 10 mM Tris, pH 7.5 Modified RIPA Lysis Buffer    10 mM Tris, pH 7.4 1% (w/v) Sodium Deoxycholate 0.1% (w/v) SDS 1% (w/v) Aprotinin 150 mM NaCl 1% (v/v) NP-40 2 mM EDTA Immunoprecipitation High Salt Wash Buffer    1.0 M NaCl 0.1% NP40 10 mM Tris, pH 7.5 RIPA Lysis Buffer    1% (v/v) Triton X-100 1% (w/v) Sodium Deoxycholate 150 mM NaCl, pH 7.5 0.1% (w/v) SDS 1% (w/v) Aprotinin Hanafusa Lysis Buffer    2.5 mM EDTA 0.1% (w/v) Sodium Deoxycholate 10 mM Sodium Phosphate, pH7.2 0.5% (v/v) NP40 1% (w/v) Aprotinin 10% (v/v) Glycerol 150 mM NaCl PBS    4.3 mM Sodium Phosphate, Dibasic (Na2HPO4) pH 7.2 2.7 mM KCl 1.8 mM Potassium Phosphate, Monobasic (KH2PO4) 137 mM NaCl Tris Buffered Saline (TBS)    Store at 4°C 150 mM NaCl 100 mM Tris, pH 7.5

BioReagents and Chemicals Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

NP-40 EDTA Glycerol Sodium Deoxycholate Tris Bromophenol Blue 2-Mercaptoethanol SDS Staph A Aprotinin Sodium Phosphate, Dibasic Potassium Chloride Sodium Chloride Potassium Phosphate Potassium Phosphate, Monobasic Bovine Serum Albumin Triton X-100

Protocol Hints Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

1. SDS (0.1 to 0.3%) may be added to the Immunoprecipitation Low Salt Wash Buffer. This will decrease the nonspecific background but may also decrease yield. 2. For analytical purposes, 50 to 75 μCi of S35-Methionine or 0.25 to 0.5 mCi of P32-Orthophosphate per 16 mm well in 0.25 ml of Methionine or Phosphate Free Media is appropriate for most proteins. For preparative purposes, a few hundred μCi of S35-Methionine in a 35 mm well and 2 to 5 mCi of P32-Orthophosphate in a 16 mm (2 mCi at most) or 35 mm (up to 5 mCi) well is appropriate. 3. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions. 4. This centrifugation may be done in a microcentrifuge with microcentrifuge tubes. However, great care must be taken not to disturb the pellet (especially with P32 samples). 5. If the samples are frozen, it is recommended that the samples be centrifuged again and the supernatant collected after thawing. 6. A titration of the antibody versus the cell number may be required (before starting a series of experiments) to determine the optimal ratio of antibody versus cell number. Generally, the antibody is in excess.

Citation and/or Web Resources Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version

1. Gilmore TD, Martin GS. Tyrosine phosphorylation of a 50K cellular polypeptide associated with the Rous sarcoma virus transforming protein pp60src. Mol Cell Biol 1982; 2:199-206 5. Pawson T, Guyden J, Kung TH, Radke K, Gilmore T, Martin GS. A strain of Fujinami sarcoma virus which is temperature-sensitive in protein phosphorylation and cellular transformation Cell 1980; 22:767-75 4. Feldman RA, Hanafusa T, Hanafusa H. Characterization of protein kinase activity associated with the transforming gene product of Fujinami sarcoma virus. Cell 1980; 22:757-65 6. Sealy L, Privalsky ML, Moscovici G, Moscovici C, Bishop JM. Site-specific mutagenesis of avian erythroblastosis virus: erb-B is required for oncogenicity. Virology 1983; 130:155-78 3. Oppermann H, Levinson Ad, Varmus HE, Levintow L, Bishop JM. Uninfected vertebrate cells contain a protein that is closely related to the product of the avian sarcoma virus transforming gene (src). Proc Natl Acad Sci U S A 1979; 76:1804-8 2. Radke K, Carter VC, Moss P, Dehazya P, Schliwa M, Martin GS. Membrane association of a 36,000-dalton substrate for tyrosine phosphorylation in chicken embryo fibroblasts transformed by avian sarcoma viruses. J Cell Biol 1983; 97:1601-11