Thiophosphate Ester Rabbit Monoclonal Antibody (Clone 51-8)

A Novel Rabbit Monoclonal for the Detection of Kinase Substrates:

• Monoclonal Antibody featured in J. Allen, et al. Nature Methods 2007 paper (6)
• Identify known and novel substrates to various kinases
• Detect context independent serine, threonine, and tyrosine phosphorylated proteins

Epitomics’ Thiophosphate Ester Rabbit Monoclonal Antibody (Cat# 2686-1, Clone 51-8) is designed to be used to detect proteins containing thiophosphate ester site following labeling as described by Allen and Shokat (Nature Methods 2007).

l. Introduction - Basic Principal
ll. Example of Kinase Substrate Detection
lll. User Manuals and Technical Info/Requirements
lV. Product Listing and Related Products
V. References

Click Here to learn more about Epitomics' RabMAbs® Technology

l. Introduction/Basic Principal

Protein phosphorlyation is the most widespread and studied regulatory signaling mechanism that occurs in both eukaryotic and prokaryotic organisms. This mechanism of adding phosphate (PO4) to a protein by a kinase forms the basis of cell signaling networks, from controlling of various protein properties (e.g. function, structure) and to being involved in all fundamental cellular processes (1, 2, 3). However, the biochemical identification of the substrates of kinase of interest (KOI) has been challenging and limited. This is due to the low abundance of most phosphoproteins, and the low affinity and substoichiometry phosphorylation of kinase-to-substrate interaction (1, 4).

To address this limitation, a bio-orthogonal affinity purification and identification of direct kinase substrates has been introduced (6). First, KOI accepts ATPγS, then it is allowed to thiophosphorylate its direct substrates. Second, the thiophospholyation site on the substrates is alkylated with p-nitrobenzylthiophosphate (PNBM). Finally, a thiophosphate ester specific rabbit monoclonal antibody (clone 51-8) is introduced to identify the tagged substrates (Figure 1). This semi-synthetic epitope tagging provides a new way to identify and isolate the substrates of various kinases (4, 5, 6).

Fig 1. Reaction sequence for affinity tagging kinase substrates

ll. Kinase Substrate Detection

Figure 2. Western Blot analysis using Thiophosphate Ester Specific RabMAb (Cat# 2686-1)*. In vitro kinase reaction using JNK1 protein (10ng) incubated with c-jun-GST substrate (1ug) using ATP-gamma-S followed by alkylation with PNBM (2.5mM). Kinase reaction was carried out for 30 minutes at room temperature. PNBM alkylation (2.5mM) was for 2 hours at room temperature. Reaction products were analyzed by western blot with thiophosphate ester specific RabMAb (Cat# 2686-1) at 1:5000, a-GST antibody confirms equal loading. Note: PNBM also reacts with cysteine residues and may decrease the affinity of other antibody-antigen interactions. *Data provided courtesy of B. Wang and Dr. K. Shokat, UCSF

Additional Data (More Kinase Substrate Detection, IP Data):
A semisynthetic epitope for kinase substrates - Allen, J.J. et al. Nature Methods, 4(6), 511-516, 2007.

III. User Manual and Techinical Information/Requirements


User Manual:

Product Name

Cat #

Clone

Datasheet

WB/IP Protocols


Technical info and Requirements:

Description: This novel rabbit monoclonal antibody (RabMAb®) can be used to detect kinase substrates containing thiophosphate esters following the kinase reaction as described by J. Allen and colleagues in Nature methods 2007. This antibody allows researchers to follow this approach to study kinase-substrate relationships of interest. The antibody is also context independent in that it can recognize serine, threonine, and tyrosine thiophosphorylated residues in a multitude of kinase phosphorylation consensus motifs.

Specificity: This antibody is specific to thiophosphorylated amino acids.

Recommended Dilutions:
WB: 1:5000
IP - Please Contact Technical Support for Details

Storage Condition/Buffer:
Store at -20 °C. Buffer: PBS with 200 mg/ml BSA, 40% glycerol and 0.02% NaN3.
Stable for 12 months from date of receipt.

Required:
PNBM (Cat# 3700-1)
ATP-gamma-S (Cat# 3701-1)
Secondary Antibody for RabMAb (Cat# 3053-1)

Optional:
N6-modified ATPgS analog

IV. Product Listing and Related Products


Rabbit Monoclonal Antibody

Product Name

Cat #

Clone

Applications

WB, IP*





Related Products

Product Name

Provider / Cat #

Clone

ATP-gamma-S*

Epitomics / 3701-1

Kinase Reaction Reagent

PNBM*

Epitomics / 3700-1

Alkylation Reagent

Goat Anti-Rabbit IgG – HRP*

Epitomics / 3053-1

Secondary for WB detection

N6-modified ATPgS analog

Biolog

Optional for Analog Specific Kinase Reaction

V. References

1. Johnson, S.A. and Hunter, T. Nature Methods 2, 17–25, 2005.
2. Hunter, T. Signaling−2000 and beyond. Cell 100, 113−127, 2000.
3. Cozzon, A.J. Ann. Rev. Microbiol. 42, 97–125, 1988.
4. Cai, D, Article Review, Mol. BioSyst., 3, 516 – 517, 2007.
5. Allen, J.J. et al. J. Am. Chem. Soc., 127(15), 5288–5289, 2005.
6. Allen, J.J. et al. Nature Methods, 4(6), 511–516, 2007. (PubMed)