| CAT No.BT-PKA-202 |
| (cAMP free) Protein Kinase A regulatory subunit-I A Recombinant, PKAR-I alpha, 25ug |
: 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 minutes 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.
Assay Conditions
Roskoski-AssayProtein kinase activity can be measured using a modified radioactive assay according to Roskoski et al.
Biological Activity
PKA regulatory subunit alpha specifically inhibits PKA catalytic subunit (Ki about 0.1nM). Activity can be restored by adding cAMP (Kact about 100nM). The binding of the catalytic subunit is dependent on the presence of ATP and Mg.
PKA regulatory subunit I a Recombinant is a dimeric 90 kDa protein.
PKAR-I alpha is purified by proprietary chromatographic techniques.
Formulation
PKA regulatory subunit-I alpha is supplied in 50% glycerol.
Physical Appearance
Sterile Filtered clear solution.
Purity
Greater than 95% as determined by SDS-PAGE.
Source
Escherichia Coli.
Storage Conditions
PKAR-Ia 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 free protein kinase type I-alpha regulatory subunit, Tissue-specific extinguisher 1, TSE1, CAR, CNC, CNC1, PKR1, PPNAD1, PRKAR1, PRKAR1A, MGC17251, DKFZp779L0468, Protein Kinase A regulatory subunit-I A, cAMP free Protein Kinase A regulatory subunit-I A, PKAR-I alpha, Regulatory (R) subunit of Protein Kinase A (PKA).
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.
