PhosphoSens Product Support 

At AssayQuant, we have validated the following plates:

• Corning 96-well Half Area White Flat Bottom Polystyrene NBS Microplate (Product Number #3642)
• Corning Low Volume 384-well White Flat Bottom Polystyrene NBS Microplate (Product Number #3824)
• ProxiPlate 384-shallow well Plus, White (Product Number #6008280)

While most of our assays use standard reaction conditions (54 mM HEPES, pH 7.5, 1 mM or KmATP, 1.2 mM DTT, 0.55 mM EGTA, 0.012% Brij-35, 10 mM MgCl2, 10 µM sensor peptide substrate, 0.05 - 10 nM kinase) we carefully optimize each assay during development to ensure that co-factors or additives are considered. For more information, please visit our Product Support Pages and look at the “Target-Specific Reaction Conditions Dropdown” or reach out to us at Support@assayqunat.com for additional information.

Yes! We see significant differences in levels of activity between enzyme vendors. When choosing a commercially available kinase preparation, an assessment of purity, specific activity, the nature and location of co-expression and purification tags (e.g., N- or C-terminal GST, His, or FLAG tags), and the size of the construct (full-length or truncated) should be considered. The most rigorous approach is to obtain a kinase from multiple sources and compare the activity with the PhosphoSens platform, where the kinetic format allows a quantitative measurement allowing the most appropriate enzyme to be selected for further study.

BSA and Brij-35 both work to stabilize the enzyme and prevent non-specific binding. Kinases at higher concentrations that are fairly stable are often ok without BSA, but we have run into some kinases that lose activity quickly when BSA is removed, especially at lower concentrations. We have removed Brij-35 as well. Some kinases are ok without it, but others lose some activity over time.

When you need high sensitivity, a filter-based reader is the way to go. Monochromator-based readers are generally less sensitive because they only transmit a small fraction of light to the sample. When the monochromator selects a wavelength, most of the light from the source is lost, causing the excitation of the fluorophore to be significantly weaker. As the emitted light passes through a series of mirrors, grating and slits, light intensity is diminished even further. From the time the initial light source flashes until the specific wavelength of light finally reaches the detector, the loss of energy is compounded, which correlates with reduced sensitivity. This means you could miss a crucial result. In contrast, filter-based readers are much more efficient at delivering light to the sample, resulting in higher sensitivity. For this reason, with a monochromator, a higher enzyme concentration may be needed.

We have titrated magnesium with a few kinases, but not all of them. The assay will tolerate up to 50 mM Mg, but the rate will likely only increase to 20 mM Mg. If increasing the Mg level, it should also be increased in the “no enzyme” blanks as well.

We routinely run the assay at 30°C. Running the assay at room temperature will slow down the reaction, and running at 37°C will speed-up the reaction. The issue with running at room temperature is that this can vary widely from lab to lab. One lab might be 25°C, while another might be 20°C. This might also vary whether it is summer or winter as well. In our labs in particular, we have seen huge variations in the lab temperature. I would expect a loss in signal at room temperature, but not necessarily “no signal”.

Contaminants that may cause an increase in signal include metal contaminants in the reagents used to make the buffers. Adding 0.55 mM EGTA to the reaction mixture can eliminate this, especially with a 30-45 minute preincubation without enzyme.