DESCRIPTION
Protein kinase B (PKB), also known as Akt, is a serine/threonine-specific protein kinase that plays a key role in multiple cellular processes such as glucose metabolism, apoptosis, cell proliferation, transcription and cell migration. The PKB kinase assay kit (Type II) is a non-radioactive, homogenous, simple, immunoblot type assay. It is designed for the assay of PKB kinase solid affinity matrices such as IP kinases or GST-PKBs on GSH beads. It is also useful for the activity analysis of PKB in the solution phase. The principle of the assay is simple. Purified PKB on the solid matrix will phosphorylate the kinase substrate-BSA conjugates (component C) in the solution, in the presence of ATP. The phosphorylated BSA-substrate is then analyzed using SDS-PAGE resolution and immunoblotting technique.
The kits were tested using PKB kinases (Akt1, 2 and 3).Sensitivity is approximately 20 ng of active GST-Akt1/assay.
Assay for activity of Akt1 using the type II kit (ICP0243)
A,250 ng; B, 125 ng; C,60 ng; D,30 ng; E, 15 ng; F, 0.00 ng
STORAGE AND STABILITY:
Stable for 6 months at 4oC from date of shipment.
KIT COMPONENTS
A. Rabbit anti-phosphoSubstrate (anti-pSubstrate): 250 μg/mL in 200 μL of Tris acetate buffer, affinitypurified rabbit polyclonal, anti-phosphopeptide substrate (RPRAApTF-NH2) antibodies (catalog# ICP0190). The anti-pSubstrate antibodies specifically recognize the phosphorylated form of the substrate (RPRAApTF-NH2) but not the non-phosphorylated form (RPRAATF-NH2). Dilute the antibody to 0.25-0.5 μg/mL with antibody dilution buffer (B) for working solution (enough to prepare 100 mL of working antibody solution).
B. 10 x Antibody Dilution Buffer (AbDB): Catalog# ICP0243b, 2x10 mL. Dilute 10 mL of AbDB to 100 mL with distilled water asworking buffer solution.
C. BSA-Substrate Conjugates (BSA-Sub): Catalog# ICP0243c, 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 kDa (enough for more than 100 assays).
D. Kinase Assay Dilution Buffer (ADB): Catalog# ICP0243d, 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# ICP0243e, 2x2 mg, Dissolve in 2 mL kinase assay dilution buffer (D) as working solution. Aliquot as 100 μL/vial and store at -20oC if not used all at once.
Additional Reagents Required but not Supplied:
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)
PROTOCOL
Stage One: Preparation of kinase
1. Preparation of Kinase in Solution: Prepare the purifiedor partially purified (fractionated) kinase PKB in 10 μL of the kinase assay dilution buffer (component D) with the required concentration in microcentrifuge tubes. For inhibitorscreening, 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-PKB (able to IP native kinase), or anti-GST, anti-HA (for tag-kinase) with 10 to 20 μL of protein A for 1 hr. Then wash away any unbound antibodies. Incubate the cell lysate in 250 μL of lysate buffer with the antibody-protein A complex in a rotor shaker at 4oC for 2 hrs. The user should select their own cell lysate buffer that contains protease and phosphatase inhibitors. The cell lysate sample should contain >20 ng of active PKB. 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..): The user should purify therecombinant kinases, such as GST-PKB, following their own protocol. 10-20 μL of affinity matrix/assay is recommended. Do a 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.
Stage Two: PKB kinase activity assay
1. Add 2.5-5 μL of the BSA-kinase substrate (component C) to the kinase sample prepared in stage one.
2. Add 5 μL of the ATP (component E) solution to initiate the kinase reaction. Incubate at 37oC 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.
Stage Three: Running SDS-PAGE and transfer;
1. Prepare a 8% SDS-acryamide gel.
2. Run the SDS-PAGE until the dye-front is approximately 2-3 cm past entering the separation gel.
3. Perform the normal gel transfer to a nitrocellulose membrane. The membrane only needs to cover between the top of separating gel and the distance to MW marker 60 kDa.
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 4oC overnight.
4. Wash the membrane with PBSt 4 times at 5 min intervals.
5. Incubate with anti-rabbit IgG secondary antibodies for 1-2 hours.
6. The user should select their own method (either colormetric or ECL system) for the signal generation
Summary of the Assay:
1. Kinase reaction: BSA-RPRAATF-NH2 + ATP + Kinase --> BSA RPRAApTF-NH2
2. SDS-PAGE resolution and transfer
3. Specific immunoblotting for detection of the kinase product BSA-RPRAApTF-NH2 probed with anti- PRAApTF-NH2 (anti-pSubstrate)
