The STK Assay Kit is based on a solid phase enzyme-linked immuno-absorbent assay (ELISA) that utilizes a specific synthetic peptide as a substrate for STK and a polyclonal antibody that recognizes the phosphorylated form of the substrate. This product is designed for testing the activity of a variety of serine/threonine kinases (STK) (see Table 1) and screening the inhibitors/activators of corresponding kinases. The kinase assay kit is non-radioactive but colorimetric.

1. 1. The principle of the assay:

1. 1. The kinase substrates are immobilized onto the 96-well plate.
   2. The kinases use ATP to phosphorylate the substrates on the plate.
   3. The phosphorylated product is detected by the phospho-substrate specific antibodies. See the product image for a visualization of the principle.

KIT COMPONENTS: The STK Assay Kit contains the following components in sufficient quantities for one 96-well plate.

Item #	Part #	COMPONENT	SIZE	DESCRIPTION
1	ICP0248-1	Substrate Microtiter Plate	96-well plate	12x8 removable strips and frame; pre-coated plate with substrate peptide for STK
2	ICP0248-2	Kinase Assay Dilution Buffer	15 mL	Buffer for the dilution of ATP, standard and samples
3	ICP0248-3	Phosphospecific Substrate Antibody	6.5 mL	Rabbit polyclonal antibody specific for phosphorylated substrate
4	ICP0248-4	Antibody Dilution Buffer	10 mL	Buffer for the dilution of Anti-Rabbit IgG: HRP Conjugate
5	ICP0248-5	Anti-Rabbit IgG: HRP Conjugate	15 µL	1 mg/mL solution of horseradish peroxidase conjugated goat anti-rabbit IgG containing 0.05% ProClin as a preservative
6	ICP0248-6	ATP	1 mg	Adenosine triphosphate
7	ICP0248-7	20X Wash Buffer	20 mL	Concentrated solution of buffer and surfactant
8	ICP0248-8	TMB Substrate	6.5 mL	Stabilized tetramethylbenzidine substrate
9	ICP0248-9	Stop Solution	6.5 mL	Acid solution to stop color reaction

The kit’s expiry date is 6 months from the date of shipping.
All reagents are stable as supplied at 4°C until the kit’s expiry date.
Unused wells of the Substrate Microtiter Plate should be resealed with the desiccant in the foil pouch provided and stored at 4°C until the kit’s expiry date.
Any remaining diluted ATP can be stored at -20°C for up to 6 months or until the kit’s expiry date, whichever is earlier.

ASSAY PROCEDURE SUMMARY

1. Bring to room temperature: Substrate Microtiter Plate, Antibody Dilution Buffer, Kinase Assay, Dilution Buffer, TMB Substrate and Stop Solution.
2. Add 60 μL samples to appropriate wells of the Substrate Microtiter Plate.
3. Initiate reaction by adding 10 μL of diluted ATP to each well.
4. Incubate for 30 minutes at 37°C.
5. Stop reaction by emptying contents of each well.
6. Wash wells ONCE with 300 μL 1X Wash Buffer.
7. Add 60 μL of Phosphospecific Substrate Antibody to each well, except the blank.
8. Incubate at room temperature for 60 minutes.
9. Wash wells 4 times with 300 μL 1X Wash Buffer.
10. Add 60 μL of diluted Anti-Rabbit IgG:HRP Conjugate to each well.
11. Incubate at room temperature for 30 minutes.
12. Wash wells 4 times with 300 μL 1X Wash Buffer.
13. Add 60 μL of TMB Substrate to each well.
14. Incubate at room temperature for 15-30 minutes. Incubation time should be determined by the investigator according to color development.
15. Add 60 μL of Stop Solution to each well.
16. Measure absorbance at 450 nm.

 

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
• The activity of the Anti-Rabbit IgG: HRP Conjugate (Part# ICP0248-3) is affected by nucleophiles such as
azide, cyanide and hydroxylamine.

The graph below illustrates results of the STK kinase assay kit using purified active STKs. Varying quantities of purified active STKs were
added the Substrate Mictotiter Plate and incubated for 30 minutes at 37°C. Activity was detected as described
in the Assay Procedure. The data represented below is an example only and should not be used to calculate
actual assay results.