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PKC Kinase Assay Kits, Type I
Catalog # ICP0212
Kits Components
1. PKC substrateplate: a 96 well plate with 12x8 removable strips. Each well waspre-immobilized with 50 μL of PKC-substrate (KRREILSRRPSYR).
2. Anti-pSubs(pCreb) antibodies (rabbit, affinity purified): 1 μg/mL, 5 mL.
3. Kinase AssayDilution buffer: 10 mL.
4. Antibodydilution buffer: 10 mL.
5. Goatanti-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. Adenosinetri-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. Peroxidasesubstrate: 10 mL, TMB (3,3'-5,5'-Tetramethylbenzidine).
8. Stop solution:10 mL.
9. 10xPBSt: 10 mL.
Kits Description
1. Qualitycontrol: The kits were tested using PKC from Sigma (#P5511). Sensitivity isapproximately 100 ng of PKC/assay.
2. Storage andStability: Stable for 6 months at 4 ℃ for date of shipment. Avoid the light and heat.
3. Descriptions: PKCis a cyclic AMP dependent protein kianse. The PKC kinase assay kit (Type I) isa non-radioactive, homogenous, simple, rapid, antigen-captured immunosorbentassay. This assay is designed for the assay of PKC in solution. The principleof the assay is simple. Purified or partially purified PKC will phosphorylatethe PKC substrate on the 96-well plate with the presence of ATP. Thephosphorylated substrate (pSubstrate) is then analyzed with theanti-phosphosubstrate antibodies. Subsequently, the binding anti-pSubantibodies will be quantified with a secondary antibody-HRP conjugates, whichgenerate the color signal from its substrate, TMB (OD at 450 nm). The finalcolor intensity is proportional to the initial PKC phosphorylation activity.
Summary of the Assay
1. Kinasereaction:
KRREILSRRPSYR +ATP+Kinase → KRREILSRRPpSYR
2. Thephosphorylated substrate on the plate will be detected with an anti-pSubstrateantibody:
KRREILSRRPSYR (noantibody binding)
KRREILSRRPpSYR(antibody binds to the plate)
3. SignalGeneration: Second antibody binds to antibody-KRREILSRRPpSYR complex formed inthe plate.
Protocol
1. Soak the wellof the substrate plate (component A) with 50 μL of kinase assay dilution buffer(component C) for 10 mins, then aspirate.
2. Prepareourified or partially purified PKC or PKC-inhibitor mixture in 30 μL of thekinase assay dilution buffer to the well of the substrate plate as prepared instep 1 (Note: For preparation of the positive control, refer to the Appendix)
3. Add samples tothe appropriate wells.
4. Add 10 μL ofthe ATP (component F) solution to the well to initiate the kinase reaction at30-35 ℃ for 90 mins. Hand-shake the strip well inevery 20 mins. If a shaker with rotate angle is applied, the speed arranged at60 rounds per min should be adequate.
5. Empty the wellthen add 40 μL of the anti-pSubstrate antibodies (component B ) at roomtemperature for 60 mins. shake the well in every 20 mins.
6. Empty the welland fill the well with 100 μL of PBSt wahsing buffer. Let the washing bufferstay for 1-2 mins then aspirate. Repeat this washing step for four times.
7. Add 40 μL ofgoat anti-rabbit IgG HRP (component E ) working soluiton to the well andincubate at room temperature for 30 mins. Shake the well in every 10 mins.
8. Empty the welland repeat the washing as step 6.
9. Add 60 μL ofTMB to develop the color (for 30-60 mins).
10. Stop thereaction 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 activityof PKC (Sigma#P5511, lot#108H7846)
Activity, transfer1 picomole of phosphate to kemptide/min/μg of protein.
Appendix
Preparation ofPositive Control
Sample Preparationof Recombinant Kinase
1. Label up 5microfuge tubes; 1-4, plus a negative control. Note: The negative controlshould only contain assay dilution buffer; no kinase.
2. Aliquot 38 μLof assay dilution into tube#1 and 30 μL into tubes 2-4.
3. Add 2 μL ofrecombinant kinase into tube#1 and mix thoroughly.
4. Transfer 10 μLfrom tube#1 to tube#2 and mix.
5. Transfer 10 μLfrom tube#2 to tube#3 and mix.
6. Transfer 10 μLfrom tube#3 to tube #4 and mix.
7. Transfer 30 μLfrom each of the mixtures (tubes 1-4 plus the control) into the appropriatewells of the substrate plate.
