Kits Components

A. Rabbit anti-pSubstrate microplate: a  96 well plate with 12 removable strips. Each well was pre-immobilized with 80  μL of anti-phosphorylated PKB/SGK-substrate antibodies (Catalog# ICP0190).  The antibodies only recognized the phosphorylated PKB/SGK substrate  (RPRAApTF-NH2) but not the non-phosphorylated substrate (RPRAATF).

B. PKB/SGK substrate, biotin conjugates:  a synthetic peptide (Biotin-RPRAATF (catlog#ICP0315). The substrate was  formulated as 250 μg/mL in 600 μL of the kinase assay dilution buffer (component  C).

C. Kinase Assay Dilution buffer: MOPS 20  mM, pH7.2; beta-Glycerolphosphate, 25 mM; EGTA, 1 mM; sodium orthovanadate,  1mM; DTT, 1 mM; MgCl2 7 mM.

D. Antibody dilution buffer: 2x10 mL;  0.5% BSA; NaCl, 1M; glycine, 0.5M, pH7.2; EDTA, 10mM; Tween20, 0.5%.

E. Avidin HRP conjugates:  Catalog#ICP9804, 1 mg/mL in PBS, 20 μL. Dilute to 1 μg/mL (1:1000 dilution)  with antibody dilution buffer (D) as working solution.

F. Adenosine tri-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.

G. Peroxidase substrate: 10 mL, TMB  (3,3'-5,5'-Tetramethylbenzidine), 250 μg/mL, in citrate buffer pH 5.0, 0.1%  H2O2. TMB is light sensitive.

Kits Description

1.Quality control: The kits were tested using PKB alpha, beta and  gamma. Sensitivity is approximately 10 ng of active GST-PKB gama/assay.

2.Storage and Stability: Stable for 6 months at 4℃ for date of shipment. Avoid the  light and heat.

3.Descriptions: The PKB kinase assay kit (Type I) is a  non-radioactive, homogenous, simple, rapid, antigen-captured immunosorbent  assay. This assay is designed for the assay of PKB purified on 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 or partial purified PKB (AKT) will phosphorylate  the PKB/SGK-substrate-biotin conjugates in the solution with the presence of  ATP. The phosphorylated substrate (pSubstrate) is then analyzed with a  sandwich ELISA. It involved the transfer of the reaction mixtures to a plate  coating with specific anti-pSubstrate antibodies. The phosphorylated  substrate-biotin in the reaction mixtures is selectively captured by the  anti-pSubstrate antibodies on the plate while the non-phosphorylated  substrate will be washed away. The residual pSubstrate-biotin on the plate  will be detected and quantified with Avidin-HRP conjugates, which generate  the color signal from its substrate, TMB (OD at 450 nm). The final color  intensity is proportional to the initial PKB phosphorylation activity.

Summary of the  Assay:

1. Kinase reaction:

Biotin-RPRAATF+ ATP+Kinase → Biotin-RPRAApTF

2. Capture of the phosphorylated substrate in the mixture in an  anti-pSubstrate plate:

Biotin-RPRAATF (wash away) and Biotin-  RPRAApTF (captured)

3.Signal Generation:

Avidin-HRP-Biotin- RPRAApTF complex  formed in the plate → color  reaction.

4. Rate of the reaction:

Avidin-HRP- Biotin- RPRAApTF complex  formed → color  intensity.

Protocol

Stage One: Preparation of kinase

1. Preparation of Kinase in Solution:  Prepare the purified or partial purified PKB in 10 μL of the kinase assay  dilution buffer (component C). For inhibitor screening, trials of serial  concentration (range from 1000 to 50 ng/20 μL) should be tested for the  optimal working concentration of kinase.

2. Preparation of the Immunoprecipitated  Kinase: Incubate anti-PKB (able to IP native kinase), or anti-GST, anti-HA  (for tag-kinase) with 10 μL to 20 μL of protein A first, approximately 1 hr.  Then wash away any unbound antibodies. Incubate the cell lysate in 250 μL of lysate buffer with a rotory  shaker, at 4℃ for 2 hrs.  Cell lysate should contains more than 10 ng of active PKB (AKT) in order to  obtain significant signal to noise. Customers should select their own cell  lysate buffer that must contain protease and phosphatase inhibitors.  Centrifuge and aspirate with PBSt twice then with kinase assay dilution  buffer (component C) twice. After aspiration, re-constitute the  immunoprecipitated matrix in 10 μL of kinase assay dilution buffer (component  C).

3. Preparation of Kinase Purified with  Affinity Matrix (GSH, Nicole, maltose): The customer should purify the  recombinant kinases such as GST-PKB following their own protocol. Recommend  10-20 μL of affinity matrix/assay. Final wash and aspiration of the matrix  with kinase assay dilution buffer Then re-constitute the aspired matrix with  10 μL of the kinase assay dilution buffer (component C).

Stage two: PKB(AKT) activity assay

1. Add 5 μL of the biotinylated kinase  substrate (component B) to kinase preparation in stage one.

2. Add 5 μL of the ATP (component F) solution to initiate the kinase  reaction at 30-35℃ for 30  mins with constant shaking or hand shake every 5 mins.

3. Stop the kinase reaction with 60 μL of  antibody dilution buffer (component D).

Stage three: Qunatitation of the kinase  phosphorylated products

1. Re-hydrate the anti-pSubstrate plate  (component A) with PBSt at room temperature for 10 mins, then wash with PBSt  twice.

2. Transfer all 80 μL of the solution of  the kinase reaction from stage two (step 3) to the correspondent well of the  anti-pSubstrate strip plate (component A).

3. Incubate at room temperature for 60  mins, and then wash the wells thoroughly with PBSt three times, at 1 min  interval.

4. Add 80 μL of the avidin-HRP (component  E, 1 μg/mL). Incubate at room temperature for 20 mins.

5. Wash the well with PBSt three times at  1 min interval, and one more time with water.

6. Add 80 μL of the HRP substrate  (component H) to each well and develop the color.

7. Read the color intensity (OD) at 370  or 655nm. or at 450 nm when stopped with 20 μL of 2N HCl

8. Calculate the relative kinase activity  as = ODsample - ODnegative

Quality Control  Test

Assay for activity of GST-PKB gamma.