Phospho-Akt Substrate (RXXS*/T*) (110B7E) Rabbit mAb (Magnetic Bead Conjugate) #8050

Immunoprecipitation for Analysis by Western Blotting

This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

1. 20X Phosphate Buffered Saline (PBS): (#9808).

2. 10X Cell Lysis Buffer: (#9803) 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 μg/ml Leupeptin

   NOTE: CST recommends adding 1 mM PMSF (#8553) before use*.

3. 3X SDS Sample Buffer: (#7722) 187.5 mM Tris-HCl (pH 6.8 at 25°C), 6% w/v SDS, 30% glycerol, 150 mM DTT, 0.03% w/v bromophenol blue
4. 6-Tube Magnetic Separation Rack: (#7017).
5. 10X Kinase Buffer (for kinase assays): (#9802) To Prepare 1 ml of 1X kinase buffer, add 100 µl 10X kinase buffer to 900 µl dH₂O, mix.
6. ATP (10 mM) (for kinase assays): (#9804) To prepare 0.5 ml of ATP (200 µM), add 10 µl ATP (10 mM) to 490 µl 1X kinase buffer.

B. Preparing Cell Lysates

1. Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
2. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold PBS.
3. Remove PBS and add 0.5 ml 1X ice-cold cell lysis buffer to each plate (10 cm) and incubate the plates on ice for 5 minutes.
4. Scrape cells off the plates and transfer to microcentrifuge tubes. Keep on ice.
5. Sonicate samples on ice three times for 5 seconds each.
6. Microcentrifuge for 10 minutes at 4°C, 14,000 x g, and transfer the supernatant to a new tube. If necessary, lysate can be stored at –80°C.

C. Immunoprecipitation

1. Take 200 μl cell lysate and add 10 µl of the immobilized antibody, incubate with rotation overnight at 4°C.
2. Wash pellet by using magnetic rack to pellet beads, then discard supernatant once completely clear and add 500 µl of 1X cell lysis buffer, vortex to resuspend and wash the beads, repeat 4 more times for a total of 5 washes.
3. Proceed to sample analysis by western blotting or kinase activity (section D).

D. Sample Analysis

Proceed to one of the following specific set of steps.

For Analysis by Western Immunoblotting

1. Resuspend the pellet with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec at 14,000 x g.
2. Heat the sample to 95–100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g.
3. Load the sample (15–30 µl) on a 4–20% gel for SDS-PAGE.
4. Analyze sample by western blot (see Western Immunoblotting Protocol).

NOTE: When using primary antibodies produced in rabbit to detect proteins with a molecular weight in the range of 50 kDa, we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (#45262) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127) as a secondary antibody to minimize interference produced by denatured rabbit heavy chain. For proteins with a molecular weight in the range of 25 kDa, Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127) is recommended to minimize interference produced by denatured mouse light chain.

When using primary antibodies produced in mouse to detect proteins with a molecular weight in the range of 50 kDa, we recommend using Rabbit Anti-Mouse IgG (Light Chain Specific) (D3V2A) mAb (HRP Conjugate) (#58802) as a secondary antibody to minimize interference produced by denatured mouse heavy chain.

For Analysis by Kinase Assay

1. Wash pellet by using magnetic rack to pellet beads, then discard supernatant once completely clear and add 500 µl of 1X kinase buffer, vortex to resuspend and wash the beads, repeat 1 more time for a total of 2 washes. Keep on ice during washes.
2. Suspend pellet in 40 µl 1X kinase buffer supplemented with 200 µM ATP and appropriate substrate.
3. Incubate for 30 min at 30°C.
4. Terminate reaction with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec.
5. Transfer supernatant containing phosphorylated substrate to another tube.
6. Heat the sample to 95–100°C for 2–5 min and microcentrifuge for 1 min at 14,000 x g.
7. Load the sample (15–30 µl) on SDS-PAGE (4–20%).