Cellular phosphorylation is a reversible, covalent modification of a protein or lipid that results in the modification of the activity of the phosphorylated molecule by inducing small conformational changes within the molecule.^1-2 Catalyzed by protein kinases, phosphate groups can be added via the transfer of the terminal phosphate from a phosphate donor, e.g. ATP to an amino acid residue, such as serine, threonine or tyrosine (Figure 1). The functions of phosphorylation, the most prevalent of post-translational modifications, include the alteration of the activity of a substrate, its subcellular localization, binding properties with other proteins.^1-2 To better understand phosphorylation and its functional role, the generation and use of phosphospecific antibodies is crucial.

Figure 1. The transfer of the terminal (γ) phosphoryl group of ATP to serine.

For raising antibodies against phosphopeptides there are 3 critical factors for success: (1) the stability of the phosphorylation, (2) the purity of the peptide and (3) post-development purification of the phosphospecific antibody. With documented experience in meeting all three factors, AnaSpec, EGT Group is a proven source for world-class phosphospecific antibody production.

With full manufacturing facilities located in Fremont, CA, AnaSpec is able to maintain ISO9001:2008 manufacturing processes and robust quality validation. Our peptide chemists routinely synthesize sequences containing single and multiple phosphoamino acids using technologies that are fine-tuned to produce high-yield, ultra-pure phosphopeptides. Antibodies generated are then substantiated to be phosphospecific.

AnaSpec offers both polyclonal and monoclonal antibody production services for the generation of phospho and non-phosphospecific antibodies. For polyclonal antibodies, researchers have a choice between two immunization protocols - the Speedy 28-day protocol (an example of results is shown in Figure 2) and the 70-day standard protocol. As a trusted provider to the global research community, we are experienced in fulfilling the most stringent demands for optimizing project success. As well, AnaSpec carries a wide selection of phosphospecific catalog antibodies such as the Anti-p53’s and Anti-TAU’s (Figures 3 & 4). Phosphospecific antibodies that are zebrafish reactive are also available.

Figure 2. The Speedy 28-day polyclonal antibody protocol was used to raise an antibody against a phosphorylated peptide. ELISA results show that the antibody had high titer and was specific towards the phosphorylated peptide.

Figure 3. Western blot analysis of non-phosphorylated (1) and phosphorylated (2) recombinant Tau proteins probed with Anti-Tau (pSer396) antibody (Cat# 54977). Only phosphorylated Tau is recognized by Anti-Tau (pSer396) (Courtesy of Dr. Nichol’s Lab, Parkinson’s Institute and Clinical Center, Sunnyvale, California).		Figure 4. Western blot analysis of non-phosphorylated (1) and phosphorylated (2) recombinant Tau proteins probed with Anti-Tau (p199/202) antibody (Cat# 54963). Only the phosphorylated Tau is recognized by Anti-Tau (pSer199/202) (Courtesy of Dr. Nichol’s Lab, Parkinson’s Institute and Clinical Center, Sunnyvale, California).

Select references to AnaSpec’s phosphopeptide antibodies:

Havel, LS. et al. (2011). Preferential accumulation of N-terminal mutant huntington in the nuclei of striatal neurons is regulated by phosphorylation. Hum Mol Genet 20, 1424. doi: 10.1093/hmg/ddr023.

Zhou, Q. et al. (2010). Characterization of in vivo keratin 19 phosphorylation on tyrosine-391. PloS one 5, e13538.

Mikels, A. et al. (2009). Ror2 receptor requires tyrosine kinase activity to mediate Wnt5A signaling. J Biol Chem 284, 30167. doi: 10.1074/jbc.M109.041715.

Zarling, AL. et al. (2006). Identification of class I MHC-associated phosphopeptides as targets for cancer immunotherapy. PNAS 103, 14889. doi: 10.1073/pnas.0604045103.

Friesner, JD. et al. (2004). Ionizing radiation–dependent γ-H2AX focus formation requires ataxia telangiectasia mutated and ataxia telangiectasia mutated and Rad3-related. Mol Biol Cell 16, 2566. doi: 10.1091/mbc.E04-10-0890.

Toivola, DM. et al. (2002). Type II keratins are phosphorylated on a unique motif during stress and mitosis in tissues and cultured cells. Mol Biol Cell 13, 1857. doi: 10.1091/mbc.01-12-0591.

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References:

1. Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002. Section 10.4, Covalent Modification Is a Means of Regulating Enzyme Activity.

2. Cohen, P. Proc R Soc Lond 234, 115 (1998). doi: 10.1098/rspb.1988.0040.