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S6K Kinase Assay Kits, Type I
Catalog # ICP0255
Kits components
A. S6K substrate plate: a 96 well plate with 12x8 removable strips.
B. Anti-pSubstrate (pS6) antibodies: 6 mL
C. Kinase Assay Dilution buffer: 12 mL
D. Antibody dilution buffer: 10 mL
E. Goat anti-rabbit IgG HRP conjugates: Catalog#ICP9804, 30 uL. Dilute to 1 ug/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).
H. Stop solution. 5 mL.
I. 10xPBSt: 10 mL
Storage and Stability
Stable for 6 months at 4oC from date of shipment. Avoid the light and heat.
Quality control
The kits were tested using p70 S6K from SignalChem. Sensitivity is approximately
Descriptions
The p70 S6K activity kit provides a safe, simple and reliable method for screening
inhibitors or activators of p70 S6K and for quantitating the activity of p70 S6K in
partially purified or purified enzyme preparations. The p70 S6K Activity Assay is based
on a solid phase enzyme-linked immunoabsorbent assay (ELISA) that utilizes a
specific synthetic peptide as a substrate for p70 S6K and a polyclonal antibody that
recognizes the phosphorylated form of the substrate. The assay is designed for
analysis of p70 S6K activity in the solution phase. In the assay, the substrate, which
is readily phosphorylated by p70 S6K, is precoated on the wells of the provided p70
S6K Substrate Microtiter Plate. The samples to be assayed are added to the
appropriate wells, followed by the addition of ATP to initiate the reaction. The kinase
reaction is terminated and a Phosphospecific Substrate Antibody is added to the wells
which bind specifically to the phosphorylated peptide substrate. The phosphospecific
antibody is subsequently bound by a peroxidase conjugated secondary antibody. The
assay is developed with tetramethylbenzidine substrate (TMB) and a color develops in
proportion to p70 S6K phosphotransferase activity. The color development is stopped
with acid stop solution and the intensity of the color is measured in a microplate
reader at 450nm.
Summary of the Assay
1) Kinase reaction:
KRRRLASLR+ ATP+active p70 S6K ---> KRRRLA(pS)LR
2) The phosphorylated substrate on the plate will be detected with an anti-pSubstrate
antibody:
KRRRLASLR
(no antibody binding)
KRRRLA(pS)LR (antibody binds to the plate)
3) Signal Generation:
2nd antibody binds to antibody-KRRRLA(pS)LR complex formed in the plate
Protocol
1. Soak the well of the substrate plate (component A) with 50 uL of kinase assay
dilution buffer (component C) for 10 min, then aspirate.
2. Prepare the assay sample (purified S6K or S6K+putatitive inhibitor mixture) in 40
uL of the kinase assay dilution buffer to the well of the substrate plate as prepared
in step 1 ( The)
3. Add samples to the appropriate wells.
4. Add 10 uL of the ATP (component F) solution to the well to initiate the kinase
o
5. Empty the well then add 50 uL of the anti-pSubstrate antibodies (component B ) at
room temperature for 60 min. Shake the well in every 20 min.
6. Empty the well and fill the well with 200 uL of PBSt washing buffer. Shake the well
and let the washing buffer stay for 1-2 min then aspirate. Repeat this washing step
for four times.
7. Add 50 uL of goat anti-rabbit IgG HRP (component E ) working soluiton to the well
and incubate at room temperature for 30 min.
8. Empty the well and repeat the washing as step 6.
9. Add 60 uL of peroxidase substrate (TMB) to develop the color (for 30-60 min) .
10. Stop the reaction with 20 uL of the stop solution (2M H2SO4)
Calculation of the relative Kinase Activity
The relative activity of the S6K = (OD(sample)-OD(negative) )
Quality Control Test
Determination the linear range of the active p70 S6K (purchased from SignalChem,
Cat#R21-10H-05)
1. Label up 5 microfuge tubes; 1-4, plus a negative control.
Note: The negative control should only contain assay dilution buffer; no kinase.
2. Aliquot 73 ul of assay dilution into tube#1 and 50 ul into tubes 2-4.
3. Add 2 ul of recombinant kinase into tube#1 and mix thoroughly.
4. Transfer 25 ul from tube#1 to tube#2 and mix.
5. Transfer 25 ul from tube#2 to tube#3 and mix.
6. Transfer 25 ul from tube#3 to tube #4 and mix.
7. Transfer 40 ul from each of the mixtures (tubes 1-4 plus the negative control) into
the appropriate wells of the substrate plate.
8. Follow the protocol to initiate the kinase reaction.
