| CAT No.BT-PKA-204 |
| cAMP-Dependent Protein Kinase A regulatory subunit-II A Recombinant, PKA-RII alpha, 3ug |
: The assay will be performed in a mixture containing 50mM MOPS (pH7.0), 10mM MgCI2, 0.25 mg/ml bovine serum albumin, 100 IJM Kemptide (peptide substrate), 100 IJM unlabeled ATP mixed with [y_32p] ATP (500-1000 cpm/pmol) and Ca subunit in a final volume of 50 IJI. Reaction is started by addition of the Ca subunit and can be stopped after a 5 minute incubation at 30¡ãC by spotting the reaction mix onto Whatman P-81 filters and soaking the filters four times in 75mM phosphoric acid (10 ml per sample) for at least 5 minutes. After four washing steps rinse filters with ethanol, dry and count.
Roskoski, R., Jr. (1983) Methods Enzymol. 99, 3-6
For the detection of phosphorylation in substrate proteins the phosphotransferase reaction can alternatively be stopped by taking aliquots of the mixture and adding SDS sample buffer. The phosphorylation status of the substrate proteins can subsequently be analysed using SDS PAGE and autoradiography.
Zimmermann, B. (1999) Journal of Biological Chemistry.274, 9, 5370-78.
Purity
Greater than 95% as determined by SDS-PAGE.
Assay Conditions
Roskoski-AssayProtein kinase activity can be measured using a modified radioactive assay according to Roskoski et al.
PKA regulatory subunit alpha specifically inhibits PKA catalytic subunit (Ki about 0.7nM). The binding is not dependent on the presence of ATPMg, however, the regulatory subunit will be phosphorylated at the binding site by PKA C alpha upon binding. Activity can be restored by adding the second messenger cAMP (Kact about 100nM). Subcellular localisation is regulated via interaction of PKA RII with so-called A-kinase anchoring proteins (AKAPs).
Description
The recombinant PKA regulatory subunit II-a is a dimeric 90 kDa protein.
Protein Kinase A is purified by proprietary chromatographic techniques.
Formulation
PKA regulatory subunit-II alpha is supplied in 50% glycerol.
Physical Appearance
Sterile Filtered clear solution.
Source
Escherichia Coli.
Stability
PKA should be stored at 4¡ãC if entire vial will be used within 2-4 weeks. For long term storage it is recommended to store at -20¡ãC.
Avoid multiple freeze-thaw cycles.
Synonyms
cAMP-Dependent Protein Kinase A regulatory subunit-II A, PKA-RII alpha.
Unit Definition
One unit is defined as the amount of recombinant PKA catalytic subunit alpha, required to incorporate 1nmol of phosphate into the specific substrate peptide kemptide (LRRASlG) in one minute at 30¡ãC.
Usage
TSZGENE's products are furnished for LABORATORY RESEARCH USE ONLY. The product may not be used as drugs, agricultural or pesticidal products, food additives or household chemicals.
