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LabChip EZ Reader MSA Assays


Overview


LabChip® EZ Reader uses a microfluidic chip technology based on a mobility shift principle. Assays utilizing fluorescently-labeled substrates are set up in polypropylene microplates. As the experiment is being run in the microplate, small volumes of each assay are "sipped" up into the LabChip microfluidic chip. The LabChip multi-sipper chips comprise small channels connected to upstream and downstream electrodes. As the reaction moves through these channels, the reaction components are separated into substrate and product (or starting material and end material) via differences in their mobility. These differences in mobility are related to the charge/mass ratio of each component (substrate, product, etc.). As each component reaches the detection window on the chip, a fluorescent peak is recorded.

ezreader_principle_ASK.jpg
Figure 1. Principle of a LabChip EZ Reader enzymatic assay. Fluorescent peptide substrate and fluorescent peptide product are sipped from a sample well of a polypropylene microplate. Separation of fluorescent product and fluorescent substrate occurs within the LabChip microfluidic chip. Separation occurs through differences in net charge of the substrate and product.

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Features


  • Microfluidic chip-based technology
  • Biochemical applications designed around a mobility-shift assay format, using fluorescently-labeled substrates
  • Can be used for mechanism-of-action (MOA) studies, real-time and end point kinetics, enzymatic profiling, screening, and hit-to-lead optimization
  • Medium throughput
  • No antibodies required
  • Compatible with many types of substrate (lipid, peptide, small molecule...)

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Applications


Enzymatic assays

  • Protein and lipid kinase assays
  • Kinase cascades
  • Protein phosphatase assays
  • Protease assays
  • Phosphodiesterase assays
  • Adenylate cyclase assays
  • ATPase assays
  • Epigenetic assays

 

Enzyme profiling assays for kinases 

  • ProfilerPro kinase selectivity assay kits

 

Binding assays

  • DNA/protein binding
  • RNA/protein binding

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Products and catalog numbers


幸运8平台彩票View a complete list of relevant products including instrumentation, chips, reagents, kits, and buffers.

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LabChip handling and preparation (best practices)


Technical brief幸运8平台彩票 describing proper LabChip multi-sipper chip storage, and how any handling issues would be manifested in your data.


Video demonstrating proper LabChip multi-sipper chip preparation.

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Creating your own substrate (substrate design)


We offer a variety of fluorecently-labeled enzymatic substrates that have been validated for use in LabChip EZ Reader assays. However, it is also possible to create your substrate or binding partner for these assays.

 

Fluorophore

The EZ Reader excites at 488 nm and measures emission at 530 nm. Fluorophores such as fluorescein (FITC), 5-FAM, and Alexa 488 have all been used as labels for LabChip EZ Reader assays. If you are creating labeled dsDNA for your assay, we recommend labeling just one strand when working with duplex DNA. This saves money, and allows you to perform competition assays to verify specificity for double-stranded target.

 

Substrate size

幸运8平台彩票It is ideal to limit the size of a peptide substrate so that there is a significant difference between the mass and/or charge of the substrate (or binding partner) and product (or binding complex). For peptide substrates, we usually recommend peptides that are 30 residues or shorter in length. We have successfully worked with peptide substrates up to 50 residues in length when necessary.

 

Net Charge

幸运8平台彩票The net charge of the substrate should be close to neutral. A net charge ranging from +3 to -3 is recommended. For peptide substrates, you may be able to alter the amino acid sequence or adjust the length of your substrate if the net charge is not within this range. It is also possible to modify the charged termini to change the net charge of your substrate.

 

Separation parameters

The Optimizer for mobility-shift assays is used to verify that a given peptide sequence can be used in a LabChip EZ Reader assay, and to systematically determine the optimal separation conditions (of voltage, pressure, and buffer sip time) for the assay. The Optimizer uses a mathematical model of the electrokinetic and pressure driven transport in the LabChip microfluidic devices to predict the resolution of product and substrate peaks for any applied voltage and pressure.

 

Further guidance

Our tech support, application, and R&D scientists are happy to provide further guidance on substrate design. If you would like to see what substrates have been tested internally for various targets, or if you would like feedback on your proposed substrate, please contact our support team.

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Tips and FAQs


Q. How do I store my LabChip multi-sipper chip?

A. After removal of the chip from the instrument, rinse the active wells 3X with deionized water and refill the wells with chip storage buffer (1 mM Di-sodium EDTA) from the chip storage jar. Store the chip in the chip container at 4°C.

 

Q. How much volume is "sipped" at a time?

A. The instrument is set up to sip 10 nL of your reaction out of the well at a time. Multiple sips can be performed from a single well at set time points, for kinetic assays.

 

Q. What is CR8?  What does it do?

幸运8平台彩票A.  Coating reagent 8 (CR8) is a positively-charged additive that can be added to the separation buffer. It will bind the silanol groups of the glass channels in the LabChip multi-sipper chip (the glass is negatively-charged) to neutralize them. In this context, non-specific interaction of substrates and products that are highly positively charged, or substrates with sequences containing repeats of a positive charge, will be reduced. 

 

Q. How do I prepare my LabChip multi-sipper chip for the assay?

A. We have created a video that shows you step-by-step how to prepare a LabChip multi-sipper chip for your assay.

 

Q. Do I need a 4-sipper or 12-sipper chip?

A. For assay development and screens, 12-sipper chips are recommended as the throughput will be higher. For ProfilerPro kinase profiling, a 4-sipper chip needs to be used because each column in the 384-plate provided in the kit contains a different substrate, and thus has a different script for the separation condition.

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Citations


 

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