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- PKA Kinase Assay Kits, Type I Kits Components 1. PKA substrate plate: a 96 well plate with 12x8 removable strips. Each well was pre-immobilized with 50 μL of PKA-substrate (KRREILSRRPSYR). 2. Anti-pSubs (pCreb) antibodies (rabbit, affinity purified): 1 μg/mL, 5 mL. 3. Kinase Assay Dilution buffer: 10 mL. 4. Antibody dilution buffer: 10 mL. 5. Goat anti-rabbit IgG HRP conjugates: Catalog#ICP9803, 0.5 mg/mL in HRP stablizing buffer, 30 μL. Dilute to 0.5 μg/mL (1:5000 dilution) with antibody dilution buffer (D) as working solution. 6. Adenosine tri-phosphate (ATP): 2 mg. Reconstitute with 2 mL kinase assay dilution buffer (C) as working solution. Once it was dissolved in the buffer, store at -20 ℃ for better stability. 7. Peroxidase substrate: 10 mL, TMB (3,3'-5,5'-Tetramethylbenzidine). 8. Stop solution: 10 mL. 9. 10xPBSt: 10 mL. Kits Description 1. Quality control: The kits were tested using PKA from Sigma (#P5511). Sensitivity is approximately 100 ng of PKA/assay. 2. Storage and Stability: Stable for 6 months at 4 ℃ for date of shipment. Avoid the light and heat. 3. Descriptions: PKA is a cyclic AMP dependent protein kianse. The PKA kinase assay kit (Type I) is a non-radioactive, homogenous, simple, rapid, antigen-captured immunosorbent assay. This assay is designed for the assay of PKA in solution. The principle of the assay is simple. Purified or partially purified PKA will phosphorylate the PKA substrate on the 96-well plate with the presence of ATP. The phosphorylated substrate (pSubstrate) is then analyzed with the anti-phosphosubstrate antibodies. Subsequently, the binding anti-pSub antibodies will be quantified with a secondary antibody-HRP conjugates, which generate the color signal from its substrate, TMB (OD at 450 nm). The final color intensity is proportional to the initial PKA phosphorylation activity. Summary of the Assay 1. Kinase reaction: KRREILSRRPSYR + ATP+Kinase → KRREILSRRPpSYR 2. The phosphorylated substrate on the plate will be detected with an anti-pSubstrate antibody: KRREILSRRPSYR (no antibody binding) KRREILSRRPpSYR (antibody binds to the plate) 3. Signal Generation: Second antibody binds to antibody-KRREILSRRPpSYR complex formed in the plate. Protocol 1. Soak the well of the substrate plate (component A) with 50 μL of kinase assay dilution buffer (component C) for 10 mins, then aspirate. 2. Prepare ourified or partially purified PKA or PKA-inhibitor mixture in 30 μL of the kinase assay dilution buffer to the well of the substrate plate as prepared in step 1 (Note: For preparation of the positive control, refer to the Appendix) 3. Add samples to the appropriate wells. 4. Add 10 μL of the ATP (component F) solution to the well to initiate the kinase reaction at 30-35 ℃ for 90 mins. Hand-shake the strip well in every 20 mins. If a shaker with rotate angle is applied, the speed arranged at 60 rounds per min should be adequate. 5. Empty the well then add 40 μL of the anti-pSubstrate antibodies (component B ) at room temperature for 60 mins. shake the well in every 20 mins. 6. Empty the well and fill the well with 100 μL of PBSt wahsing buffer. Let the washing buffer stay for 1-2 mins then aspirate. Repeat this washing step for four times. 7. Add 40 μL of goat anti-rabbit IgG HRP (component E ) working soluiton to the well and incubate at room temperature for 30 mins. Shake the well in every 10 mins. 8. Empty the well and repeat the washing as step 6. 9. Add 60 μL of TMB to develop the color (for 30-60 mins). 10. Stop the reaction with 20 μL of the stop solution (2 N HCL). Calculation of the relative Kinase Activity OD(smaple)-OD(negative)/ng of kinase Quality Control Test Assay for activity of PKA (Sigma#P5511, lot#108H7846) Activity, transfer 1 picomole of phosphate to kemptide/min/μg of protein. Appendix Preparation of Positive Control Sample Preparation of Recombinant Kinase 1. Label up 5 microfuge tubes; 1-4, plus a negative control. Note: The negative control should only contain assay dilution buffer; no kinase. 2. Aliquot 38 μL of assay dilution into tube#1 and 30 μL into tubes 2-4. 3. Add 2 μL of recombinant kinase into tube#1 and mix thoroughly. 4. Transfer 10 μL from tube#1 to tube#2 and mix. 5. Transfer 10 μL from tube#2 to tube#3 and mix. 6. Transfer 10 μL from tube#3 to tube #4 and mix. 7. Transfer 30 μL from each of the mixtures (tubes 1-4 plus the control) into the appropriate wells of the substrate plate.
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