Epitomics, Inc - The Rabbit Monoclonal Antibody Company

Protocols

Protocols: Immunoprecipitation

Immunoprecipitation Protocol

1. Solutions and reagents

1.1 10X Cell Lysis Buffer (Epitomics Cat. #: 3500-1)

1.2 Add Phenylmethylsulfonyl fluoride (PMSF) to the Cell Lysis buffer to a final concentration of 1mM. (Stock solution 100mM PMSF). Note: Add fresh before each use

1.3 Goat Anti-Rabbit IgG HRP-labeled secondary antibody
(Epitomics cat# 3053-1 recommended).

2. Preparation of Non-denaturing Cell Lysate

2.1. Adherent cells

2.1.1 Grow cells in a T175 flask to ~90% confluency.

2.1.2 Wash flask twice with cold PBS.

2.1.3 Add 3 ml of 1x Cell lysis buffer containing 1mM PMSF to the T175 flask.

2.1.4 Transfer cell lysates to Eppendorf tubes and sonicate for 10-15 seconds.

2.1.5 Spin at 14,000 rpm, 4 °C for 10 minutes.

2.1.6 Aliquot the supernatant into a new microcentrifuge tubes and store at -80 °C.

2.2. Suspension cells

2.2.1 Harvest and count cells.

2.2.2 Wash cells twice with cold PBS.

2.2.3 Add 30 μl of 1x Cell lysis buffer containing 1mM PMSF to 1 x 10 6 cells

2.2.4 Sonicate for 10-15 seconds.

2.2.5 Spin at 14,000 rpm, 4 °C for 10 minutes.

2.2.6 Aliquot the supernatant into a new microcentrifuge tubes and store at -80 °C.

3. Immunoprecipitation Protocol

3.1. Mix 200-300 μl of non-denatured cell lysate with appropriate dilutions of primary antibody.

3.2. Incubate overnight at 4 °C with gentle rocking.

3.3. Add 25 μl of Protein G beads (50% bead slurry) and incubate 2 hours at 4 °C with gentle rocking.

3.4. Microcentrifuge for 30 seconds and discard the supernatant. Wash pellet 3 times with cold PBS.

3.5. Resuspend the pellet with 20 μl of 2x SDS reducing sample buffer containing β-mercaptoethanol (5%, v/v) and heat the sample at 95-100 °C for 3 minutes.

3.6. Briefly centrifuge the sample.

3.7. Load 16 μl of sample on to a SDS-PAGE gel or freeze the sample for later use (reboiled for 3 minutes prior loading onto the SDS-PAGE gel).

3.8.Perform Western blotting analysis using HRP-conjugated anti-rabbit IgG as secondary antibody (Epitomics cat#3053-1). Reccomended Western Blot Protocol.

4. Optional Immunoprecipitation Protocol (for target antigens with a size of ~25 or ~50 kDa)

4.1. Mix 200–300 μl of precleared cell lysate with primary antibody.

4.2. Incubate overnight at 4 °C with gentle rocking.

4.3. Add 25 μl of Anti-Rabbit IgG Beads and incubate 2 hours at 4°C with gentle rocking.

4.4. Microcentrifuge for 30 seconds, and remove the supernatant. Wash pellet 3 times with cold 1XPBS.

4.5. Resuspend the pellet with 20 μl 2x SDS reducing sample buffer containing 2-mercaptoethanol and heat the sample at 95–100 °C for 3 minutes.

4.6. Briefly centrifuge the sample.

4.7. Load 16 μl of sample on SDS-PAGE gel or freeze the sample for later use (reboiled for 3 minutes prior to SDS-PAGE).

4.8. Perform Western blotting analysis using TrueBlot HRP-conjugated anti-rabbit IgG as secondary antibody (1:1,000 eBiosciences Cat. #18-8816-33), which preferentially detects the non-reduced form of rabbit IgG over the reduced, SDS-denatured form of IgG.