Protein kinase C also known as PKC is a family of protein kinase enzymes that are involved in controlling the function of other proteins through the phosphorylation of hydroxyl groups of serine and threonine amino acid residues on these proteins. PKC enzymes in turn are activated by signals such as increases in the concentration of diacylglycerol (DAG) or calcium ions (Ca2+). Hence PKC enzymes play important roles in several signal transduction cascades. The PKC kinase assay kit (Type II) is a non-radioactive, homogenous, simple, immunoblot type of assay. This type of assay is designed for purified kinase on solid affinity matrices such as IP kinases or PKCs on GSH beads. It is also useful for the activity analysis of PKC in the solution phase. The principle of the assay is that the PKC kinase phosphorylate the substrate-BSA conjugates (component C) in the presence of ATP. The phosphorylated BSA-substrate is then analyzed using SDS-PAGE resolution and immunoblotting technique.

The kit is stable for 6 months at 4°C from the date of the shipment.

1. PREPARATION OF SUBSTRATE MICROTITER PLATE
   1. Determine the number of wells to be used. If less than 96 pre-coated microtiter wells are needed, remove the excess wells from the frame and return them to the foil pouch. Reseal the pouch containing the unused wells with the desiccant and store at 4°C.
2. ADDITION OF STANDARDS AND SAMPLES
   1. Add 60 μL of each of the following to appropriate wells:
      1. Purified Active PKC
      2. Samples (previously prepared, see page 6-7)
      3. Blank (Kinase Assay Dilution Buffer with no kinase)
      4. Negative Control (Inhibitor/Activator Diluent with no inhibitor or activator) (use for inhibitor or activator screening studies)
   2. Initiate reaction by adding 10 µL of diluted ATP (previously diluted, see page 6) to each well, except the blank. To avoid cross contamination, change pipette tips for each well.
   3. Cover wells with an adhesive plate sealer or plastic wrap and incubate at 37°C for 30-60 minutes, preferably with gentle shaking.
      NOTE: It is recommended that the experiment use the predetermined time point generated during the initial experiment as outlined in the RECOMMENDATIONS PRIOR TO USING THE ASSAY SECTION on page 5.
   4. Stop reaction by emptying contents of each well. Invert the plate and carefully pat dry on clean paper towels.
3. WASHING
   1. Remove liquid from all wells.
   2. Add 300 μL of 1X Wash Buffer to all wells, using a multi-channel pipette, manifold dispenser, automated microplate washer, or a squirt bottle. (To reduce background, it may be necessary to soak the wells for 30-60 seconds between each wash).
   3. Remove liquid from all wells. Repeat the removing and washing 3 more times with 1X Wash Buffer for a total of 4 washes.
   4. After the 4th wash, remove the liquid from all wells. Invert the plate and carefully pat dry on clean paper towels.
4. ADDITION OF PHOSPHOSPECIFIC SUBSTRATE ANTIBODY
   1. Add 60 μL of the Phosphospecific Substrate Antibody to each well, including the blank.
   2. Cover wells with a fresh adhesive plate sealer (or plastic wrap) and incubate at room temperature for 60 minutes, preferably with gentle shaking.
   3. Wash plate as described in Step 3.
5. ADDITION OF ANTI-RABBIT IgG: HRP CONJUGATE (previously diluted, see page 7)
   1. Add 60 μL of the previously diluted Anti-Rabbit IgG:HRP Conjugate to each well, including the blank.
   2. Cover wells with a fresh adhesive plate sealer (or plastic wrap) and incubate at room temperature for 30 minutes, preferably with gentle shaking.
   3. Wash plate as described in Step 3.
6. ADDITION OF TMB SUBSTRATE AND ACID STOP SOLUTION
   1. Add 60 μL of the TMB Substrate to each well.
   2. Incubate the plate at room temperature for 15-30 minutes (incubation time should be monitored by the investigator according to color development).
   3. Add 60 µL of the Stop Solution to each well in the same order that the TMB Substrate was added.
7. MEASURING ABSORBANCE
   1. Set up the microplate reader according to the manufacturer’s instructions.
   2. Set wavelength at 450 nm.
   3. Measure the absorbance.

The kits were tested using recombinant PKC. Sensitivity is approximately 5 ng of active /assay under un-optimized conditions.

Assay for activity of kinase (ng/assay) using the PKC kit (ICP0211)