Catalog# ICP0216
KIT COMPONENTS
1. PKA substrate plate: a 96 well plate with 12x8 removable strips. Each well was pre-immobilized with 50μL of PKA-substrate (KRREILSRRPSYR).
2. Anti-pSub (pCreb) antibodies (rabbit, affinity purified): 1μg/mL, 5mL.
3. Kinase Assay Dilution buffer: 10mL.
4. Antibody dilution buffer: 10mL.
5. Goat anti-rabbit IgG HRP conjugates: Catalog# ICP9803, 0.5mg/mL in HRP stabilizing buffer, 30μL. Dilute to 0.5μg/mL (1:5000 dilution) with antibody dilution buffer (D) as working solution.
6. Adenosine tri-phosphate (ATP): 2mg. Reconstitute with 2mL kinase assay dilution buffer (C) as working solution. Once it is dissolved in the buffer, store at -20oC for better stability.
7. Peroxidase substrate: 10mL, TMB (3,3'-5,5'-Tetramethylbenzidine).
8. Stop solution: 10mL.
9. 10x PBSt: 10mL.
KIT DESCRIPTION
Quality control: The kits were tested using PKA from Sigma (#P5511). Sensitivity is approximately 100ng of PKA/assay.
Storage and Stability: Stable for 6 months at 4oC from date of shipment. Avoid the light and heat.
Descriptions: PKA is a cyclic AMP dependent protein kinase. 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 the secondary antibody-HRP conjugate, which generates 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: Secondary antibody binds to antibody-KRREILSRRPpSYR complex formed on the plate.
PROTOCOL
1. Soak the well of the substrate plate (component A) with 50μL of kinase assay dilution buffer (component C) for 10 mins, then aspirate.
2. Prepare purified or partially purified PKA or PKA-inhibitor mixture in 30μL 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 10μL of the ATP (component F) solution to the wells to initiate the kinase reaction at 30-35oC for 60 mins. Hand-shake the strip 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 wells then add 40μL of the anti-pSubstrate antibodies (component B) at room temperature for 60 mins. Shake every 20 mins.
6. Empty the wells and fill the well with 100μL of PBSt washing buffer. Let the washing buffer stay for 1-2 mins then aspirate. Repeat this washing step four times.
7. Add 40μL of goat anti-rabbit IgG HRP (component E) working solution to the well and incubate at room temperature for 30 mins. Shake the wells every 10 mins.
8. Empty the wells and repeat the washing as step 6.
9. Add 60μL of TMB to develop the color (for 30-60 mins).
10. Stop the reaction with 20μL of the stop solution (2 N HCl).
11. Calculation of the relative kinase activity: OD (sample)-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 5 microfuge tubes; 1-4, plus a negative control. Note: The negative control should only contain assay dilution buffer; no kinase.
2. Aliquot 38μL of assay dilution into tube#1 and 30μL into tubes 2-4.
3. Add 2μL of recombinant kinase into tube#1 and mix thoroughly.
4. Transfer 10μL from tube#1 to tube#2 and mix.
5. Transfer 10μL from tube#2 to tube#3 and mix.
6. Transfer 10μL from tube#3 to tube #4 and mix.
7. Transfer 30μL from each of the mixtures (tubes 1-4 plus the control) into the appropriate wells of the substrate plate.
