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Protein Ser/Thr Phosphatase (PSP) Assay System |
 | | | | Protein Ser/Thr Phosphatase (PSP) Assay System |
| | | Discontinued as of 4/1/09. Contact info@neb.com for assistance. | |
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This product is not available for online ordering.
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Description:
The Protein Phosphatase Assay Systems are designed to assay serine/threonine (1) or tyrosine (2) protein phosphatase activity. The Protein Serine/Threonine Phosphatase (PSP) Assay System and the Protein Tyrosine Phosphatase (PTP) Assay System include the commonly-used model protein substrate, Myelin Basic Protein (MyBP), which can be used to assay type 1 and 2 protein serine/threonine phosphatases, tyrosine protein phosphatases and dual specificity protein phosphatases.
The 32P (or 33P)-labeled MyBP is prepared by phosphorylation on serine and threonine residues with cAMP-dependent Protein Kinase (PKA) (3) (PSP Assay System) or by phosphorylation on tyrosine residues with Abl Protein Tyrosine Kinase (Abl) (4) (PTP Assay System) in the presence of [γ-32P or 33P] ATP.
Protein serine/threonine phosphatase or protein tyrosine phosphatase activity can then be determined by measuring the release of inorganic phosphate (TCA-soluble 32P-radioactivity) from 32P-labeled MyBP. Protein Phosphatase 1 (PP1) (PSP Assay System) (5) or T-Cell Protein Tyrosine Phosphatase (TC PTP) (PTP Assay System) (6) are included as positive controls.
Phosphoamino acid analysis of MyBP phosphorylated by PKA using PSP Assay System (A) and MyBP phosphorylated by Abl using PTP Assay System (B). MyBP was treated with the kinase in the presence of [γ-33P] ATP, TCA-precipitated phosphoprotein was subjected to partial acid hydrolysis. Phosphoamino acids were separated by two-dimensional thin layer electrophoresis, internal standard markers were stained with ninhydrin, and 33P-labeled phosphoamino acids were detected by autoradiography.
Advantages:- Unique: These are the only Protein Phosphatase Assay Systems employing Myelin Basic Protein (MyBP). The Protein Tyrosine Phosphatase (PTP) Assay System is the only kit available that allows the high sensitivity detection of protein tyrosine phosphatases.
- Sensitive: By using radiolabeled substrates, the highest degree of sensitivity is achieved. As little as a few pmol of released phosphate can be detected.
- Universal: Myelin Basic Protein (MyBP) is phosphorylated on multiple serine/threonine and tyrosine residues, thereby constituting a broad specificity substrate.
Kit Components:
10% Brij 35
ATP
cAMP-dependent Protein Kinase (PKA), catalytic subunit
MnCl2 (10X)
PKA Reaction Buffer (10X)
PP1 Reaction Buffer Pack (10X)
Protein Phosphatase 1 (PP1)
Substrate Solubilization Buffer
Storage Conditions
Storage Temperature:
-70°C
Notes
General notes:- Labeled nucleotide is not included in the Protein Phosphatase Assay Systems.
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
The PSP Assay System is functionally tested using PP1, PP2A, and λ-PPase, a dual specificity protein phosphatase.
The PTP Assay System is functionally tested using protein tyrosine phosphatases such as LAR, TC PTP, YOP, and a dual specificity protein phosphatase, λ-PPase.
Reagents Sold Separately
cAMP-dependent Protein Kinase (PKA), catalytic subunit
PKA Reaction Buffer
Protein Phosphatase 1 (PP1)
Companion Products
Protein Tyr Phosphatase (PTP) Assay System
Various concentrations of PP1 were reacted with 10 µM 33P-Ser/Thr-MyBP (with respect to incorporated phosphate; MyBP was phosphorylated by PKA with incorporation of ~3 mol phosphate/mol using the PSP Assay System) under standard reaction conditions. Reactions were terminated by addition of TCA, and TCA-soluble radioactivity (inorganic phosphate released from the substrate) was measured (A). The linear range of dephosphorylation of 33P-Ser/Thr-MyBP by PP1 from the same experiments is shown in Panel B.
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