Invitrogen™ Z'-LYTE™ Kinase Assay Kit - Tyrosine 2 Peptide

Complete lack of an assay window can either be a problem with the instrument setup or with the development reaction. To determine whether or not the problem is with the development reaction or with the instrument setup, please do the following:

Using buffer to make up the volume from reagents that are not used, perform a development reaction as follows.

- 100% Phosphopeptide control: Do not expose the 100% phosphopeptide to any development reagent, this will ensure that it is not cleaved and will give the lowest value of the ratio.
- Substrate: Expose the 0% phosphopeptide, the substrate, to 10-fold higher development reagent than necessary according to the Certificate of Analysis (COA). This will ensure full cleavage after 1 hour and will give the highest value of the ratio.

Typically, for properly developed Z'-LYTE reactions, there is a 10-fold difference in the ratio of the 100% phosphorylated control and the substrate. If not, the dilution of the development reagent used needs to be checked. Please refer to the Certificate of Analysis (COA) for your kit and lot. Please note that the Ser/Thr 7 phosphopeptide is easy to over develop.

If no difference in ratios is observed, either the reagents are very over- or under-developed, or, more likely, it is an instrument problem. Please refer to our instrument setup guides in our instrument compatibility portal (http://www.thermofisher.com/instrumentsetup). If your instrument is not listed there, please contact Drug Discovery Technical Support at drugdiscoverytech@thermofisher.com.

Here is an example using ERB2 (HER2) kinase. You will see information that looks like this:

ERBBE (HER2)
- The 2X ERBB2 (HER2) / Tyr 06 mixture is prepared in 50 mM HEPES, pH 7.5, 0.01% BRIJ-35, 10 mM MnCl2, 1 mM EGTA, 2 mM DTT, 0.02% NaN3.
- The final kinase reaction consists of 1.78 - 30.4 ng ERBB2 (HER2) and 2 µM Tyr 06 in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 5 mM MgCl2, 5 mM MnCl2, 1 mM EGTA, 1 mM DTT, 0.01% NaN3.
- After the 1 hour kinase reaction, 5 µL of a 1:128 dilution of Development Reagent A is added.
Please note that the ERBBE (HER2) kinase requires Mn2+ for activity, and hence the kinase is not prepared in 1X Kinase Buffer A (50 mM HEPES, pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA).

The amount of kinase to use will be similar to what we show here depending on the lot number of the kinase and the ATP level that you select. Note that the 1.78-30.4 ng is the amount of kinase in 10 µL, not a concentration. Do a small titration of the kinase to target 30% phosphorylation of the Z'-LYTE substrate. Your ng usage should be similar to what we show.

Here is the premise of the above outline from SelectScreen. This is a variation on the customer protocol.

1. 4X compounds are prepared in 1X Kinase Buffer A after the serial dilution is done at 100X in 100% DMSO.
2. (2X Kinase/2X Substrate) is prepared at 2X in the kinase buffer. For ERBB2, the kinase buffer can be made from scratch or by spiking a concentrated form of the additives into 1X Kinase Buffer A. For ERBB2 kinase, this means addition of Mn2+, DTT and NaN3. Also, please see options below*.
3. 4X ATP is prepared in 1X Kinase Buffer A.

Add to the assay plate:

- 2.5 µL of 4X compound.
- 5 µL of 2X Kinase/2X Substrate
- 2.5 µL of 4X ATP to start the reaction

The buffer used to prepare the kinase substrate mixture is 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MnCl2, 1 mM EGTA, 2 mM DTT and 0.02% NaN3. *There are a couple of options.
- Make the buffer from scratch.
- Spike in the additives to 1X Kinase Buffer A as follows. Note that we make the buffer from scratch, so this is just a mathematical example.

To 1 mL of 1X Kinase Buffer A add:
- 2 µL of 5,000 mM MnCl2
- 2 µL of 1,000 mM DTT
- 2 µL of 10% NaN3

Then use this buffer to prepare the 2X kinase/2X substrate mixture.

After 1 hour, perform the kinase development reaction. Use the dilution outlined in the Certificate of Analysis (COA) that can be found on the product page, for the lot number of the kit purchased.

Please refer to the Z'-LYTE Screening Protocol and Assay Conditions (http://assets.thermofisher.com/TFS-Assets/LSG/brochures/20101112_SSBK_Customer_Protocol_and_Assay_Conditions.pdff.pdf) for the following information:

-The approximate amount of kinase required
-If additives or changes are needed for Kinase Buffer A
-The ATP Km apparent value for the assay
-The identity of the control inhibitor and its IC50 value