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MOLECULAR BIOLOGY: WORKING WITH DNA

PURIFICATION: IMMUNOPRECIPITATION

Immunoprecipitations of [³⁵S] Metabolically-labeled Cell Extract

Immunoprecipitations of [35S] Metabolically-labeled Cell Extract Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Contributor:	The Laboratory of Donald Rio at the University of California, Berkeley
Overview Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
This protocol assumes that the user has already made an extract from cells labeled with [35S]. Employ the IgG antibody of choice for the immunoprecipitation.
Procedure Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
A. Incubation of Cell Extract with Antibody 1. Set up the following immunoprecipitation reactions on ice:    50 μl of Buffer A    1 μl of antibody or buffer as a negative control    5 μl of [35S] labeled-cell extract (about 1 million cpm---See Protocol ID#913. CAUTION! See Hint #1.) 2. Incubate the reaction at 0°C for 4 hr or overnight (on ice or in a cold room). B. Preparation of Staphylococcus aureus (Staph A) 1. Prepare Staph A by thawing a tube of Staph A on ice. Mark the volume on the tube. 2. Sonicate the Staph A three times for 30 sec each burst. 3.Centrifuge the sample for 30 sec in a microcentrifuge and discard the supernatant. 4. Resuspend the pellet in 300 μl of Buffer A with a Pasteur pipette. 5. Centrifuge the sample for 30 sec in a microcentrifuge and discard the supernatant. 6. Repeat Steps #4 through #5 with Buffer A one more time. 7. Resuspend the Staph A pellet in Buffer A back to its original volume and store at 4°C for no more than one month. C. Incubation of Extract with Staph A 1. Add 12 μl of freshly washed Staph A from Step #7 to each immunoprecipitation reaction from Step #A1. 2. Incubate the reactions for 1 hr on ice or in a cold room. 3. Centrifuge for 30 sec in a microcentrifuge and discard the supernatant. 4. Wash the pellet by resuspending it in 300 μl of Tris/LiCl at 4°C. Vortex to mix (see Hint #2). 5. Centrifuge for 30 sec in a microcentrifuge and discard the supernatant. Skip to step #D11 if only one antibody incubation is required. D. Second Antibody Incubation 1. Resuspend the pellet in 36 μl of PBS/SDS to release the protein-antibody complex from the Staph A cells. 2. Incubate for 1 hr on ice or in a cold room. 3. Centrifuge for 30 sec in a microcentrifuge,transfer the supernatant to a fresh tube, and discard the pellet. 4. Add 40 μl of Buffer A/BSA and 1 μl of Secondary Antibody. 5. Incubate at 0°C for 4 hr or overnight on ice in a cold room. 6. Wash more Staph A from Step #B7 in Buffer A by centrifuging for 30 sec in a microcentrifuge and resuspending the pellet in Buffer A. Wash the Staph A once more. 7. Add 12 μl of Staph A to each of the immunoprecipitation reactions from Step #D5. 8. Incubate for 1 hr on ice or in a cold room. 9. Centrifuge for 30 sec in a microcentrifuge and discard the supernatant. 10. Wash the pellet with 300 μl of Tris/LiCl and centrifuge for 30 sec (see Hint #2). 11. Resuspend the Staph A pellets in 12 μl of 2X Sample Buffer and boil them for 5 min. 12. Centrifuge for 30 sec and load the supernatants onto a polyacrylamide gel (see Protocol ID#455).
Solutions Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Secondary Antibody    Antibody of choice Primary Antibody    Antibody of choice PBS/SDS    1% (w/v) SDS pH 7.2 2.7 mM KCl 4.3 mM Sodium Phosphate Dibasic (Na2HPO4) 1.8 mM Potassium Phosphate Monobasic (KH2PO4) 137 mM NaCl Sample Buffer (2X)    10% (v/v) 2-Mercaptoethanol 6% (w/v) SDS 20% (v/v) Glycerol 100 mM Tris-Cl, pH 6.8 Buffer A/BSA    0.5% (v/v) Igepal CA 630 50 mM Tris-HCl, pH 8.0 120 mM NaCl 10 mg/ml Bovine Serum Albumin (BSA) Tris/LiCl    0.5% (v/v) Igepal CA 630 50 mM Tris-HCl, pH 8.0 200 mM LiCl Buffer A    0.5% (v/v) Igepal CA 630 50 mM Tris-HCl, pH 8.0 120 mM NaCl
BioReagents and Chemicals Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Sodium Chloride Bovine Serum Albumin SDS Igepal CA 630 Primary Antibody Secondary Antibody Potassium Phosphate, Monobasic Sodium Phosphate, Dibasic 2-Mercaptoethanol Glycerol Tris-HCl Lithium Chloride Potassium Chloride
Protocol Hints Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
1. This substance is a biohazard. Please consult this agent's MSDS for proper handling instructions. 2. The optimal number of washes must be empirically determined and depends on the amount of extract, the abundance, stability, and accessibility of the protein/antigen of interest, and the specificity of the primary antibodies, among other variable factors.