PKA Kinase Assay Kits, Type I(ICP0216)

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96 Assays/Kit
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  • Product Description

    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.
  • Product Specific References

    1. Scientific Reports. 2016. 6: 34374: 1-11. doi: 10.1038/srep34374
    2. PLoS Genetics. 2019. 15(5). doi: 10.1371/journal.pgen.1008176
    3. Stemcell Research & Therapy. 2021. 12(1):269. doi: 10.1186/s13287-021-02325-6
    4. Antioxidants.2021.10(2):184. doi: 10.3390/antiox10020184
  • 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 90 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

    Assay for activity of PKA (Sigma#P5511, lot#108H7846)
    Activity, transfer 1 picomole of phosphate to kemptide/min/μg of protein.

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