Why is my background fluorescence increasing over time?

Evaporation from wells, especially with long kinetic reads under low humidity conditions, can be significant. Without a plate seal, the reaction mixture will evaporate, and the baseline fluorescent signal will increase. Plates should be sealed with an optically clear adhesive film that still allows top-reading with minimal light scattering. We recommend DOT Scientific T480 Plate Seals.

Does maximum RFU for the same substrate vary by kinase?

The maximum RFU for a given Sensor Peptide Substrate is constant. Kinases exhibit different levels of activity, catalytic efficiency, and stability. We recommend performing a kinase titration and systematically testing buffer components (e.g., add BSA or glycerol) to rule out variables including enzyme instability.

Are all assays performed under the same reaction conditions?

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.

Does enzyme source matter?

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.

Can you exclude both BSA and Brij-35 in the reaction conditions?

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.

How can I address low Signal to Background (S:B)?

The concentration of kinase used may be too low or the kinase is unstable or insufficiently activated. Kinase may need to be pre-activated and/or additional co-factors may be required for the kinase to achieve full activity, e.g., Ca(II) or lipid for PKCs, Ca(II)/Calmodulin for some CAMKs. An activating kinase may also be used if it doesn't phosphorylate the Sox-based substrate. It may be necessary to titrate the ATP and/or Mg(II) to determine the concentration that results in the maximum fluorescence increase, which can be peptide specific. Generally, a 1.5-fold increase in fluorescence upon phosphorylation is all that is required to achieve a robust Z' value of >0.8. Some samples may contain compounds that interfere with fluorescence and/or activity measurements in this assay. It is advisable to run a background fluorescence scan prior to kinetic data acquisition. Below is a list of known compounds for which the indicated concentration results in < 10% inhibition of the PhosphoSens signal (higher concentrations should be avoided): CaCl2, 2.5 mM; Detergents (0.01% SDS, 1% Triton X-100); DMSO, 10%; DTT, 5 mM; EDTA, 1 mM or EGTA, 2 mM; MnCl2, 250 µM can be used for Mn(II)-dependent kinases (250x physiological levels); NaCl, 150 mM; Na3VO4, 40 µM; β-glycerophosphate, 10 mM.

My instrument has a monochromator, not filter cubes. Are there any changes I need to make?

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.

What is the upper limit of magnesium concentration?

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.

Have we ever run assays at different temperatures to better understand the difference in activity? Could running at RT be a significant contributor to an absence of signal?

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”.

What contaminants may be in buffers/reagents that may cause these non-linear artifacts?

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.

Why am I seeing less fluorescence at high ATP concentrations?

For some of the kinase titrations we do start to see inhibition at 500-1000 µM ATP. When determining ATP Km, we don’t use these points. Too high an ATP concentration either inhibits the reaction itself or results in product inhibition. Some enzymes are more sensitive to this than others.

Is DTT or TCEP more suitable for characterizing covalent? And can you replace DTT with TCEP?

Yes, you can replace DTT with TCEP! TCEP is better for characterizing covalent, Cys reactive derivatives because it does not contain thiols like DTT does. Some covalent inhibitors also contain thiols so DTT might compete for binding, while TCEP would not!