Product Pathways - Motif Antibodies

Phospho-(Ser/Thr) Kinase Substrate Antibody Sampler Kit #9920

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Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  E-P=ELISA (Peptide)
Reactivity Key:  H=Human  M=Mouse  R=Rat  All=All  Mk=Monkey  Dr=Drosophila

Specificity / Sensitivity

Each antibody detects endogenous levels of phospho-(Ser/Thr) proteins of specific kinase substrate groups.Phospho-(Ser/Thr) Akt Substrate (110B7E) Rabbit mAb preferentially recognizes peptides and proteins containing phospho-serine/threonine preceded by arginine at positions -5 and -3, in a manner largely independent of the surrounding amino acid sequence. Some cross-reactivity is observed for peptides that contain phospho-serine/threonine preceded by arginine at position -3 . No cross-reactivity is observed with the corresponding nonphosphorylated sequences or with other phospho-serine/threonine-containing motifs. By ELISA, the antibody recognizes a wide range of peptides with phospho-threonine or Phospho-serine with arginine at -3 and -5 position.Phospho-(Ser) PKC Substrate Antibody detects endogenous levels of many cellular proteins only when phosphorylated at serine residues surrounded by Arg or Lys at the -2 and +2 positions and a hydrophobic residue at the +1 position. The antibody does not cross-react with nonphosphorylated serine residues, with phospho-threonine in the same motif, or with phospho-serine in other motifs.Phospho-PKA Substrate (RRXS*/T*) (100G7E) Rabbit mAb detects peptides and proteins containing a phospho-Ser/Thr residue with arginine at the -3 and -2 positions. It is a useful tool in identifying new substrates of PKA. The antibody recognizes other -3 arginine-bearing phospho-Ser/Thr peptides, such as substrate motifs for Akt and PKC, to a lesser extent. It does not recognize the nonphosphorylated substrate motif peptides.Phospho-(Thr) MAPK/CDK Substrate Mouse mAb detects phospho-threonine only when followed by proline. It reacts with proteins/peptides phosphorylated on the Thr-Pro motif in an otherwise highly context-independent fashion. The antibody does not cross-react with phospho-threonine in the absence of an adjacent proline. The antibody does not cross-react with phospho-tyrosine, but does react with some phospho-serine peptides containing the phospho-serine-proline motif (e.g., phospho-Elk-1).Phospho-(Ser) CDKs Substrate Antibody detects phospho-serine in a (K/R)(S*)PX(K/R) motif. The antibody is phospho-specific but does not recognize phospho-serine in the absence of the CDK motif. The antibody does not cross-react with phospho-threonine- or phospho-tyrosine-containing peptides/proteins.Phospho-(Ser/Thr) ATM/ATR Substrate Antibody detects endogenous levels of proteins containing the ATM/ATR substrate motif. This antibody preferentially binds peptides and proteins that contain phospho-Ser/Thr preceded by Leu or similar hydrophobic amino acids at the -1 position and followed by Gln at the +1 position. The antibody does not cross-react with corresponding nonphosphorylated sequences or with other phospho-Ser/Thr-containing motifs.

Western Blotting

Western blot analysis of whole cell lysates of Jurkat cells untreated and treated with PMA (50 ng/ml) and ionomycin (1 µM) for 20 minutes prior to lysis. Proteins were separated by 2D electrophoresis prior to blotting and probed with Phospho-(Ser) PKC Substrate Antibody #2261.

Western Blotting

Western blot analysis of whole cell lysates from Jurkat cells untreated and treated with 1 µg/ml nocodazole for 12 hours prior to lysis, using Phospho-(Thr) MAPK/CDK Substrate mAb #2321. Proteins were separated by 2D electrophoresis prior to blotting.

Western Blotting

Western blot analysis of extracts from HeLa cells either untreated or treated with the microtubule destabilizing agents nocodazole or paclitaxol, using Phospho-(Ser) CDKs Substrate Antibody #2324.

Western Blotting

Chk2 transfected and UV treated COS cell extracts immunoprecipitated with Chk2 antibody then detected by Western blotting using Phospho-(Ser/Thr) ATM/ATR Substrate Antibody #2851.

Western Blotting

Western blot analysis of extracts from serum-starved NIH/3T3 cells, phosphorylated in vitro with Akt kinase, or treated in culture with PDGF or FBS/Calyculin A, using Phospho-(Ser/Thr) Substrate (110B7E) Rabbit mAb #9614.

Western Blotting

Western blot analysis of extracts from serum-starved A431 cells, phosphorylated in vitro with PKA kinase or treated in culture with forskolin/IBMX, using Phospho-PKA Substrate (RRXS*/T*) (100G7E) Rabbit mAb #9624. Lysis buffer: 1.0% Triton X-100 (lanes 1 and 2), 2.0% SDS (lanes 3 and 4).

ELISA-Peptide

Phospho-(Ser) PKC Substrate Antibody #2261 DELFIA® assay: Signal-to-noise ratio of phospho- versus nonphospho-peptides. (S* denotes phosphorylated serine.) (DELFIA® is a registered trademark of PerkinElmer, Inc.)

ELISA-Peptide

Signal to noise ratio of ELISA reading, using Phospho-(Thr) MAPK/CDK Substrate mAb #2321 with phospho-Thr-Pro containing peptides (T*P) and nonphospho-Thr-Pro containing peptides. (T* denotes phosphorylated threonine.)

ELISA-Peptide

Phospho-(Ser) CDKs Substrate Antibody #2324 DELFIA® Assay: Signal-to-noise ratio of phospho- versus nonphospho-peptides. (S* denotes phosphorylated serine.) (DELFIA® is a registered trademark of PerkinElmer, Inc.)

ELISA-Peptide

Phospho-(Ser/Thr) ATM/ATR Substrate Antibody #2851 DELFIA® assay: Signal-to-noise ratio of phospho- versus nonphospho-peptides. (S* or T* denote phosphorylated serine or threonine.) (DELFIA® is a registered trademark of PerkinElmer, Inc.)

ELISA-Peptide

Phospho-(Ser/Thr) Akt Substrate (110B7E) mAb #9614 ELISA assay: Signal-to-noise ratio of phospho- versus nonphospho-peptides. (T* and S* denote phosphorylated threonine and serine.)

ELISA-Peptide

Phospho-PKA Substrate (RRXS*/T*) (100G7E) Rabbit mAb #9624: Signal-to-noise ratio of phospho- versus nonphospho-peptides. (T* and S* denote phosphorylated threonine and serine.)

Source / Purification

Polyclonal antibodies are produced by immunizing rabbits with phospho-peptides containing the kinase substrate motif and purified by protein A and peptide affinity chromatography.Rabbit monoclonal antibodies are produced by immunizing rabbits with synthetic phospho-peptides (KLH-coupled).Mouse monoclonal antibody (isotype: IgM) is produced by immunizing mice with a phospho-(Thr) MAPK/CDK substrate motif-containing peptide and is purified by protein A chromatography.

Background

Phospho-(Ser/Thr) kinases and phosphatases play critical roles in a wide range of biological processes. Each phospho-(Ser/Thr) kinase phosphorylates serine or threonine within a specific motif. Akt phosphorylates substrates at a serine or threonine only in a conserved motif characterized by arginine at positions -5 and -3 (1). Conventional PKC isozymes phosphorylate substrates containing serine or threonine, with arginine or lysine at the -3, -2 and +2 positions, and a hydrophobic amino acid at position +1 (2,3). A consensus phosphorylation site of PKA is serine or threonine with arginine at the -2 and -3 positions (3). The MAPK and CDK families of serine/threonine protein kinases phosphorylate threonine or serine followed by proline residue (3-5).The consensus amino acid sequence for CDK substrate is (K/R)(S*)PX(K/R), where denotes any one of the 20 amino acids and S* is the phosphorylation site (6-8). ATM and the related kinase ATR phosphorylate serine or threonine in an S*/T*Q motif (9,10).Antibodies specific to particular kinase substrates are invaluable reagents in determining kinase activity and identifying potential new kinase substrates. CST has developed antibodies that recognize phosphorylated serine or threonine within the context of a protein motif that is phosphorylated by Akt, PKC, PKA, MAPK/CDK, CDKs or ATM/ATR. As shown by peptide pairing ELISA, each phospho-(Ser/Thr) kinase substrate antibody in this sampler kit is specific to its kinase substrate motif.

1. Alessi, D.R. et al. (1996) FEBS Lett. 399, 333-338.
2. Nishikawa, K. et al. (1997) J. Biol. Chem. 272, 952-960.
3. Pearson, R.B. and Kemp, B.E. (1991) Methods Enzymol. 200, 62-81.
4. Karin, M. (1994) Curr. Opin. Cell Biol. 6, 415-424.
5. Lewis, T.S. et al. (1998) Adv. Cancer Res. 74, 49-139.
6. Songyang, Z. et al. (1996) Mol. Cell Biol. 16, 6486-6493.
7. Songyang, Z. (1999) Prog. Biophys. Mol. Biol. 71, 359-372.
8. Holmes, J.K. and Solomon, M.J. (1996) J. Biol. Chem. 271, 25240-25246.
9. Kastan, M.B. and Lim, D.S. (2000) Nat. Rev. Mol. Cell Biol. 1, 179-186.
10. Zhao, H. and Piwnica-Worms, H. (2001) Mol. Cell Biol. 21, 4129-4139.

Application References

• Xu, W. et al. (2003) The heat shock protein 90 inhibitor geldanamycin and the ErbB inhibitor ZD1839 promote rapid PP1 phosphatase-dependent inactivation of AKT in ErbB2 overexpressing breast cancer cells. Cancer Res. 63, 7777-7784. This article references the use of Phospho-(Ser/Thr) Kinase Substrate Antibody Sampler Kit in the following applications: Western Blotting

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License/Use Restrictions: Use of CST Motif Antibodies within certain methods (e.g., U.S. Patent No.'s 7,198,896 & 7,300,753) may require a license from CST. For information regarding academic licensing terms please have your technology transfer office contact CST Legal Department at CST_ip@cellsignal.com. For information regarding commerical licensing terms please contact CST Business Development at cbunker@cellsignal.com.