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PhosphoSens-Kinetic Kinase Assay

Reaction setup and protocols

PhosphoSens-Red  Kinase Assay

Reaction setup and protocols

PhosphoSens-Kinetic Phosphatase Assay

Reaction setup and protocols

Plate Reader Setup Guide

General and instrument setup guidelines

Frequently Asked Questions

General and troubleshooting

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Reach out to our team for additional information or support

PLATE READER SETUP

General Plate Reader Setup Guidelines

PhosphoSens delivers rich information using a simple add-and-read workflow and a standard fluorescence microplate reader. Below, we provide general instrument setup guidelines and downloadable Plate Reader Specific Guides. 

PhosphoSens-Kinetic General Plate Reader Setup

  • Use an instrument capable of reading fluorescence intensity in kinetic (continuous) mode at an excitation wavelength of ~360 nm (358-363 nm) and an emission wavelength of ~492 nm (485-498 nm). 

  • Readings can be made at your desired intervals and duration. We read every 30 seconds for 4 hours (or until assay plateaus).

  • All PhosphoSens Assays are developed and optimized to be run at 30°C.

  • Place your plate in the reader, and select a well to use for adjusting gain and focus. To adjust the gain, use a positive control (phosphopeptide) to avoid going off scale during the assay. When finished, select "Start Adjustment" (or equivalent) to calculate its optimal focal height and make any gain adjustments necessary.

PhosphoSens-Red General Plate Reader Setup

  • Use an instrument capable of reading an endpoint time-resolved fluorescence (RFU) reading with an excitation wavelength of ~360 nm and an emission wavelength  of ~620 nm. 

  • After the kinase reaction is complete, and Europium has been added, return your plate to the reader without it's plate seal, adjust the gain and read.

Plate Reader-Specific Setup Guides

For additional instrument-specific setup support, contact our support team.

INSTRUMENT SETUP GUIDE

BMG LABTECH PHERAstar/PHERAstarPlus /PHERAstar FS/PHERAstar FSX Microplate Readers Assay Setup Guide

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FAQ

Frequently Asked Questions

General Questions

How many assays can I run per 1 mg of Sensor Peptide?

PhosphoSens Sensor Peptides range in molecular weight (MW) based on the size and sequence of the substrate. The average MW or a sensor peptide is 2,150 g/mol. This size Sensor Peptide will be re-suspended in approximately 450 µL of diluent. If running an assay in a 384-well plate, with a final well volume of 20 µL and Sensor Peptide concentration of 10 µM, you can run 2,250 assays!

How do I resuspend my lyophilized sensor peptide?

Reference the "Solubilization" section of the printed Certificate of Analysis (CofA) that is printed and shipped with every order. If you do not see this, please contact us at support@assayquant.com and we will share an electronic copy of your CofA. 

How should I store my sensor peptide?

Lyophilized sensor peptides can be stored up to 2 years at -20°C or below.

Once resuspended, sensor peptides should be aliquoted and stored at -20°C for up to 1 year. Minimize thaw/freeze cycles for best stability.

What plates should I use?

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)

How do I place an order?

Orders can be placed using the following methods:

1. If you know what you'd like to order, contact your Area Business Manager or  orders@assayquant.com.

2. If you are a new customer or have any questions, please contact us at hello@assayquant.com.

Additional Resources

Looking for more? Check out our Resources for all the latest information.

Kinome Tree Mapping Application

Visualize, explore, and customize your kinome profiling results with our NEW Kinome Tree Mapping Application!

Troubleshooting Questions

Low Signal to Background?

Reasons you may be seeing low signal to background include:
  1. The kinase concentration may be too low, the kinase is unstable, or insufficiently activated. The 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.

  2. It may be necessary to titrate the ATP and/or Mg(II) to determine the concentration resulting in the maximum fluorescence increase, which can be peptide specific. Generally, a 1.5-fold increase in fluorescence upon phosphorylation is sufficient to achieve a robust Z' value of >0.8).

  3. Some samples may contain compounds that interfere with fluorescence and/or activity measurements in this assay. You may need 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 (used for Mn(II)-dependent kinases  and corresponds to 250x physiological levels)
• NaCl, 150 mM
• Na3VO4, 40 µM
• β-glycerophosphate, 10 mM

Maximum RFU for the same substrate varies by kinase?

The maximum RFU for a given Sox-substrate is constant. If you see changes when using the same substrate with different kinases, this indicates that the kinase may be unstable under the conditions tested. Do a kinase titration and systematically test buffer components (e.g., add BSA or glycerol). You may also need to rule out
product inhibition.

Different Sox-based substrates have different maximum RFU?

This is expected, each individual Sox-based substrate may have a different maximum RFU as this value is influenced by the sequence of the peptide.

Plateau with constant RFU is observed

A plateau with constant RFU may indicate that sufficient time has elapsed for the enzyme to completely phosphorylate all the substrate.

Alternatively, the kinase being used is not stable under the conditions being tested. This may require you to to optimize conditions or testing kinase preparations from different suppliers.

Using a chemically synthesized phosphopeptide corresponding to the substrate provides a control to show the maximum RFU that should be achieved.

Background fluorescence increases over time?

An increase in background (all reaction components without enzyme) over time may be caused by evaporation from wells, especially with long kinetic reads or under low humidity conditions. Plates should be sealed with an optically-clear adhesive film that still allows top-reading with minimal light scattering. We recommend PerkinElmer TopSeal-A Plus (6050185) applied with either a roller or a paddle (VWR 60941-118 or 60941-128, respectively).

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