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REACTIVITY SENSITIVITY MW (kDa) Isotype
H M R Mk Endogenous 37 Rabbit IgG
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Immunoprecipitation

Immunoprecipitation of extracts from 293 cells using GβL (86B8) Rabbit mAb (Magnetic Bead Conjugate) (lane 1). Rabbit (DA1E) mAb IgG XP® Isotype Control (Magnetic Bead Conjugate) #8726 was used as a negative control (lane 2). The western blot was probed using GβL (86B8) Rabbit mAb #3274 and detected with Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb #3678 followed by Anti-mouse IgG, HRP-linked Antibody #7076.

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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*.

  1. 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
  2. 6-Tube Magnetic Separation Rack: (#7017).
  3. 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.
  4. 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: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (L57A3) mAb (#3677) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).

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%).

posted December 2007

protocol id: 408

Product Usage Information

Application Dilutions
Immunoprecipitation 1:20

Storage: Supplied in PBS Buffer (pH 7.2), 0.1% Tween® 20. Store at 4°C. Do not aliquot the antibodies.

Specificity / Sensitivity

GβL (86B8) Rabbit mAb (Magnetic Bead Conjugate) detects endogenous levels of total GβL protein.


Species Reactivity: Human, Mouse, Rat, Monkey

Source / Purification

GβL (86B8) Rabbit mAb is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gln210 of human GβL.

Product Description

This Cell Signaling Technology (CST) antibody is immobilized by the covalent reaction of hydrazinonicotinamide-modifed antibody with formylbenzamide-modified magnetic bead. GβL (86B8) Rabbit mAb (Magnetic Bead Conjugate) is useful for immunoprecipitation assays of GβL protein. The unconjugated GβL (86B8) Rabbit mAb #3274 reacts with human, mouse, rat, and monkey GβL protein. CST expects that GβL (86B8) Rabbit mAb (Magnetic Bead Conjugate) will also recognize GβL in these species.


Cell growth is a fundamental biological process whereby cells accumulate mass and increase in size. The mammalian Target of Rapamycin (mTOR) pathway regulates growth by coordinating energy and nutrient signals with growth factor-derived signals (1). mTOR is a large protein kinase with two different complexes. One complex contains mTOR, GβL, and raptor, which is a target of rapamycin. The other complex, insensitive to rapamycin, includes mTOR, GβL, and rictor (1). GβL associates with the kinase domain of mTOR and stimulates mTOR kinase activity (2). A reduction in GβL expression has been shown to decrease in vivo phosphorylation of S6K1 (2).


1.  Sarbassov, D.D. et al. (2004) Curr Biol 14, 1296-302.

2.  Kim, D.H. et al. (2003) Mol. Cell 11, 895-904.


Entrez-Gene Id 64223
Swiss-Prot Acc. Q9BVC4


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 5,675,063.