Recombinant human IRR protein (ab69988)

Thank you for your recent telephone enquiry.

I am sorry to confirm, as we discussed on the telephone, that ab69988 Insulin Receptor R protein has not been tested and guaranteed in binding assays, and has not been tested with ab123768 insulin protein.

The Insulin receptor R protein has been tested in a kinase assay, and I ahve copied the protocol for you below.

I am sorry we do not have the testing data you require on this occasion. However, I hope you will find this information useful and if you have any further questions, please do not hesitate to contact me.

Active Kinase Assay ab69988 Insulin Receptor R antibody

Active IRR (0.1ug/ul) diluted with Kinase Dilution Buffer IV and assayed as outlined in sample activity plot.
(Note: these are suggested working dilutions and it is recommended that the researcher perform a serial dilution of Active IRR for optimal results).

Kinase Assay Buffer II diluted at a 1:4 ratio (5X dilution) with final 50ng/ul BSA solution.
Kinase Assay Buffer II
Buffer components: 25mM MOPS, pH 7. 2, 12.5mM beta-glycerol-phosphate, 20mM MgC12, 12.5mM MnC12, 5mM EGTA, 2mM EDTA. Add 0.25mM DTT to Kinase Assay Buffer prior to use.
[33P]-ATP Assay Cocktail
Prepare 250 mM [33P]-ATP Assay Cocktail in a designated radioactive working area by adding the following components: 150l of 10mM ATP Stock Solution, 100 ul [33P]-ATP (1mCi/100ul), 5.75ml of Kinase Assay Buffer II . Store 1 ml aliquots at –20oC
10 mM ATP Stock Solution
Prepare ATP stock solution by dissolving 55mg of ATP in 10ml of Kinase Assay Buffer II. Store 200ul aliquots at –20oC.
Substrate
Axltide synthetic peptide substrate (KKSRGDYMTMQIG) diluted in distilled H2O to a final concentration of 1mg/ml.

Assay Protocol
Step 1. Thaw [33P]-ATP Assay Cocktail in shielded container in a designated radioactive working area.
Step 2. Thaw the Active IRR, Kinase Assay Buffer, Substrate and Kinase Dilution Buffer on ice.
Step 3. In a pre-cooled microfuge tube, add the following reaction components bringing the initial reaction volume up to 20ul:
Component 1. 10ul of diluted Active IRR (Catalog #I07-11G-10)
Component 2. 5ul of 1mg/ml stock solution of substrate (Catalog #A16-58)
Component 3. 5ul distilled H2O (4oC)
4. Set up the blank control as outlined in step 3, excluding the addition of the substrate. Replace the substrate with an equal volume of distilled H2O.
5. Initiate the reaction by the addition of 5 ul [33P]-ATP Assay Cocktail bringing the final volume up to 25ul and incubate the mixture in a water bath at 30oC for 15 minutes.
6. After the 15 minute incubation period, terminate the reaction by spotting 20 ul of the reaction mixture onto individual pre-cut strips of phosphocellulose P81 paper.
7. Air dry the pre-cut P81 strip and sequentially wash in a 1% phosphoric acid solution (dilute 10 ml of phosphoric acid and make a 1L solution with distilled H2O) with constant gentle stirring. It is recommended that the strips be washed a total of 3 intervals for approximately 10 minutes each.
8. Count the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation counter.
9. Determine the corrected cpm by removing the blank control value (see Step 4) for each sample and calculate the kinase specific activity as outlined below.

Calculation of [P33]-ATP Specific Activity (SA) (cpm/pmol)

Specific activity (SA) = cpm for 5 ul [33P]-ATP / pmoles of ATP (in 5 ul of a 250 mM ATP stock solution, i.e., 1250 pmoles)

Kinase Specific Activity (SA) (pmol/min/ug or nmol/min/mg)

Corrected cpm from reaction / [(SA of 33P-ATP in cpm/pmol)*(Reaction time in min)*(Enzyme amount in ug or mg)]*[(Reaction Volume) / (Spot Volume)]