Protein kinase Calso known asPKC is a family ofprotein kinaseenzymesthat are involved in controlling the function of otherproteinsthrough thephosphorylationofhydroxyl groups ofserineandthreonineamino acid residues on these proteins. PKC enzymes in turn are activated by signals such as increases in the concentration ofdiacylglycerol(DAG) orcalciumions (Ca²⁺). Hence PKC enzymes play important roles in severalsignal transduction cascades. The 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 kits were tested using recombinant PKC.Sensitivity is approximately 5 ng of active /assay under un-optimized conditions.

A.Rabbit anti-phosphoSubstrate (anti-pSubstrate): 250 µg/mL in 200 µL of Tris acetate buffer, affinitypurified rabbit polyclonal, anti-phosphopeptide substrate (pCrebtide) antibodies (catalog#ICP0180). The anti-pSubstrate antibodies recognize specifically the phosphorylated form of the substrate but not the non-phosphorylated substrate. Dilute the antibody to 0.25-0.5 µg/mL with antibody dilution buffer (B) for working solution (enough for preparation of 100 mL of working antibody solution).

B.10 x Antibody Dilution Buffer (AbDB): Catalog#ICP0211b, 2x10 mL. Dilute 10 mL of AbDB to 100 mL with distilled water asworking buffer solution.

C.BSA-Substrate Conjugates (BSA-Sub): Catalog#ICP0211c, 250 µg/mL in 500 µL kinase assay dilutionbuffer (D). Approximately 10 molecules of kinase substrate peptide (RPRAATF-NH2) were conjugated to one molecule of BSA. Apparent molecular weight of the major conjugate is 70-80KD. The apparent MW for the aggregated form is approximately 150 kd (enough for more than 100 assays).

D.Kinase assay Dilution Buffer (ADB): Catalog#ICP0211d, 10 mL. 20 mM MOPS, pH7; 25 mM beta-Glycerolphosphate; 1 mM EGTA; 1 mM sodium orthovanadate; 1 mM DTT; and 2 mM MgCL.

E.Adenosine Triphosphate (ATP): Catalog#ICP0211e, 2x2 mg, Dissolve in 2 mL kinase assay dilution buffer (D) as working solution. Aliquot as 100 µL/vial and stored at -20^oC if not used it all in one time.

1. Anti-rabbit Ig"s secondary antibodies;

2. PBSt washing buffer;

3. 3% BSA or 3% skimmed milk as blocking buffer

4. All reagents and materials for SDS-PAGE, transfer materials, and signal generating system (such as BCIP/NBT or ECL)

1.Preparation of Kinase in Solution: Prepare the purifiedor partially purified (fractionated) kinase in 10 µL of the kinase assay dilution buffer (component D) with proper concentration in microcentrifuge tubes. For inhibitor screening, serial dilutions (range from 1000 to 50ng/assay) of kinase should be tested for the optimal working concentration.

2.Preparation of the Immunoprecipitated Kinase: Incubate anti-PKC (able to IP native kinase), or anti-tag (for tag-kinase) with 10 to 20 µL of protein C for 1 hr. Then wash away any unbound antibodies. Incubate the cell lysate sample in 250 µL of lysate buffer with the antibody protein A complex in a rotor shaker, at 4^oC for 2 hrs. The users should select their own cell lysate buffer that should contain protease and phosphatase inhibitors. Cell lysate sample should contains >20 ng of active PKC. Centrifuge and aspirate with PBSt twice then with kinase assay dilution buffer (D) twice. After aspiration, re-constitute the immunoprecipitated matrix in 10 µL of kinase assay dilution buffer (D).

3.Preparation of Kinase Purified with Affinity Matrix (GSH, Ni, maltose, etc.): The users should purify therecombinant kinases such as PKC following their own protocol. Recommend 10-20 µL of affinity matrix/assay. Final wash and aspiration of the matrix with kinase assay dilution buffer (D) then re-constitute the aspired matrix with 10 µL of the kinase assay dilution buffer.

1. Add 2.5-5 µL of the BSA-kinase substrate (component C) to kinase sample prepared in stage one.

2. Add 5 µL of the ATP (component E) solution to initiate the kinase reaction at 37^oC for 60 min with constant shaking or hand shake every 5-min.

3. Stop the kinase reaction with 20 µL of SDS-sample loading buffer and boil for 2 min.

1. Prepare a 8% SDS-acryamide gel.

2. Run the SDS-PAGE until the dye-front is approximately 2-3 cm after entering the separation gel.

3. Perform the normal gel transfer to nitrocellulose membrane. The membrane needs only to cover between the top of separating gel and the distance to MW marker 60 kd.

Stage Four: Immunoblotting.

1. Block the membrane with 3% BSA or 3% skimmed milk for 30-60 min

2. Wash the membrane twice with PBSt.

3. Incubate with 10 mL of anti-pSubstrate (component A), 0.25-0.5 µg/mL in antibody dilution buffer (component B) at room temperature for 2-4 hours, or 4^oC overnight.

4. Wash the membrane with PBSt 4 times at 5 min interval.

5. Incubate with anti-rabbit IgG secondary antibodies for 1-2 hours.

6. The users should select their own methods (either colormetric or ECL system) for the signal generation.

1. Kinase reaction: BSA-KRREILSRRPSYR+ATP+Kinase->BSA-RREILSRRPpSYR

2. SDS-PAGE resolution and transfer.

3. Specific immunoblotting for detection of the kinase product BSA-KRREILSRRPpSYR probed with anti-pSubstrate antibody