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Enzymatic Assays Using LabChip EZ Reader

 

Overview


LabChip® EZ Reader microfluidic technology has been used for a variety of enzymatic assays including protein and lipid kinase, protease, phosphatase, phosphodiesterase, methyltransferase/demethylase, and acetyltransferase/deacetylase assays. This technology can be used for screening assays, real-time kinetic assays, end-point assays, and mechanism-of-action studies. Profiling studies can also be performed (for more information, refer to our Kinase profiling application page). A variety of substrates can be utilized, from small molecule substrates to lipid substrates to peptide substrates. Kinase cascade assays have also been demonstrated.

Enzymatic reactions using fluorescent substrates are first set up in polypropylene microplates. Aliquots from each reaction are sipped up into the LabChip microfluidic multi-sipper chip. Separation of fluorescent substrate and fluorescent product occurs within the chip via a mobility difference, where substrate or product (depending on substrate design and instrument parameters) moves more quickly through the channel based on the charge. For example, phosphorylation of a substrate imparts a negative charge on the product. The difference in net charge on the substrate and phosphorylated product allows separation as both are pulled through the microfluidic chip. The two species (substrate and product) migrate at different rates through the microfluidic chip.

ezreader_enzymatic_1_ASK.jpg
Figure 1. LabChip EZ Reader technology

The end result is a trace showing separate peaks for fluorescent product and fluorescent substrate over time. Because both substrate and product are being monitored, the specific activity of the enzyme and enzyme reaction rates can be determined.

ezreader_substrate_product_peak_ASK.jpg
Figure 2. Sketch of output from run. In this example, fluorescent product moved faster through the chip and reached the Detection window first. Separate peaks are seen for fluorescent product and fluorescent substrate. Percent conversion from substrate to product can be determined using the peak heights.

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What do I need to run this assay?


Available from PerkinElmer:

  • Fluorescently-labeled substrate (refer to Products and catalog numbers section, our kinase substrate selection guide, or you can >make your own FITC, 5-FAM, or Alexa Fluor® 488 labeled substrate)
  • LabChip multi-sipper chip (Cat. No. 760404 for 12-sipper chip, Cat. No. 760394 for 4-sipper chip)
  • Separation buffer with 1X coating reagent 8 (Cat. No. 760367)
  • LabChip EZ Reader instrument

 

Available from other suppliers:

  • Enzyme
  • Reaction buffer (make sure to include any cofactors required for your enzymatic reaction; we also recommend adding 0.015% Brij-35 to your reaction buffer to prevent nonspecific binding)
  • Polypropylene microplate - our applications team uses 384-well plates from Costar® (Cat. No. 3656), or shallow-well 384-well plates from Seahorse Biosciences (Cat. No. 201288-100). 384-well plates must be used with our 12-sipper chips, due to the spacing of the sippers. 96-well or 384-well polypropylene plates may be used with our 4-sipper chips. 

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Products and catalog numbers


View a complete list of relevant products including instruments, chips, reagents, kits, and buffers. 

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Protocol-in-brief


ezreader_binding_pib4_ASK.jpg
Protocol-in-brief for enzymatic assay

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Assay development


View our detailed assay development guide, which includes step-by-step instructions, sample plate maps, example data, and data processing information. Each assay must be optimized individually. Listed below are the recommended assay development steps for LabChip enzymatic assays.

  1. Substrate selection. In many cases, you can find an appropriate substrate for your enzyme using our Substrate selection guide. If you are planning to label your own substrate, please refer to our section on Substrate design.
  2. Enzyme titration/time course experiment
  3. Substrate titration
  4. Cofactor titration
  5. Inhibitor titration.

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Setting up your assay


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Tips and FAQs


Q. How many times can I re-use my LabChip multi-sipper chip?

A. You should be able to get 50,000 data points (assays) for our 12-sipper chips, or 16,500 data points (assays) for our 4-sipper chips.  

 

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. Can I develop my own substrate to use in the assay?

A. Yes, you can design your own substrate. Please refer to our section on substrate design. Keep in mind that we do offer validated substrates as part of our catalog for many target enzymes.

 

Q. What kind of substrates can I use?

A. You can use any substrate in our catalog, as well as substrates that are designed according to our recommendation. Scientists have published the use of fluorescent peptide, nucleic acid, lipid, and small-molecule substrates with our technology. The overall (net) charge of the substrate/product should be preferentially in the range of -3 to +3. Very positive substrates will have a non-specific interaction with the glass surface in the multi-sipper chip.

 

Q. What is CR8?  What does it do?

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. Is there anything in my reaction buffer that could interfere with the separation?

A. Most reaction buffer components will work fine. Assays have been run in HEPES, MOPS, and Tris buffers. There is some indication that MES buffer may cause issues - we recommend avoiding MES if possible. Low to moderate salt concentrations should not cause issues. For detergents, we recommend using Brij-35 (final concentration 0.015%), Tween, or Triton™ X-100. We recommend avoiding detergents like SDS, which can create bubbles. 

Problems with buffer components may be encountered if the ionic strength of the reaction buffer differs dramatically from that of our Separation buffer. Our Separation buffer is a HEPES-based buffer at pH 7.3, and contains 0.015% Brij.

  • If you must run your assay in a buffer that has a pH below 6 (for example a low-pH citrate or glycine buffer), you may need to alter our Separation buffer to match your reaction buffer more-closely.
  • High concentrations of salt can potentially cause issues by minimizing the separability of product and substrate by charge shielding effects that occur at high ionic strength, increasing the buffer conductivity (and therefore current) and thereby limiting the voltages that can be obtained by the instrument, and increasing the rate at which buffer is depleted due to the higher currents. High salt effects can be mitigated by diluting your terminated reaction with Separation buffer.
  • If you are doing an end-point assay and will be stopping your enzymatic reaction with EDTA or an inhibitor, we recommend adding the EDTA or inhibitor directly to an aliquot of Separation buffer, and using this as your Stop Solution. If you will be using acid to stop your reaction, you may need to flood the reaction with Separation buffer before sipping. Because a stop buffer is not used in kinetic runs, care must be taken to ensure that large buffer mismatches between the reaction and Separation buffer do not adversely affect the separation of product and substrate. Contact our support teams for further guidance.

 

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, 4-sipper 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.

 

Q. Do I have to run my assay in polypropylene plates?

A. Proteins and peptides can stick to polystyrene plastic. Because you will be sipping up molecules from the plate wells, the actual concentration of the peptides sipped is affected by the loss of material to the well surface. For this reason, we recommend using polypropylene plates for your enzymatic assay. 

 

Q. Where can I find the tech sheet, with the instrument parameters, for my substrate?

A. If you would like to view a tech data sheet, please contact our support team.

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Mechanism-of-action studies


View more information on mechanism-of-action studies using LabChip EZ Reader technology. Understanding the mechanism of inhibition for a lead compound can be vital to determination of structure-activity relationships and the success of a drug development program. With the real-time kinetics capability of the EZ Reader, generation of reaction progress curves from single reaction wells is straightforward. The platform returns high quality data, as concentrations of both substrate and product are measured at each time point. A wide range of substrate, co-factor, and compound concentrations can be accommodated, allowing flexibility in reaction setup for characterization of inhibitor effects on enzyme activity.   

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Application notes, posters, guides, and other resources


  • Assay development guide: Step-by-step instructions, plate maps, and sample data for developing your assay
  • Kinase substrate selection guide: Table showing recommended ProfilerPro substrates for various kinases.
  • Recommended book: Robert A. Copeland, "Evaluation of Enzyme Inhibitors in Drug Discovery: A Guide for Medicinal Chemists and Pharmacologists (Methods of Biochemical Analysis)". Link to PubMed.
  • Application note: Histone Deacetylases: Substrate and Inhibitor Profiling using Microfluidic Mobility-Shift Detection. Assays were optimized for enzymes from three HDAC classes; substrate profiling with histone and non-histone substrates; real-time deactylation rates; inhibitor testing (IC50 and rank potency)
  • Application note: LabChip Mobility-Shift Assay - Phosphodiesterases.    
  • Poster: Microfluidic Mobility-Shift Assays for Mechanistic Studies with HDAC inhibitors. Kinetics of HDAC3 and HDAC6 activity in presence of different inhibitors; MOA.   
  • Poster: Selectivity Profiling and Mechanistic Characterizaton of Phosphodiesterase Inhibitors Using a Microfluidic Assay. A panel of 13 PDE assays was developed to determine inhibitor potency and selectivity; mechanism of action studies including reversibility and mode of inhibition.
  • Poster: Characterization of HDAC Substrates and Inhibitors using a Microfluidic Mobility-Shift Assay. Design of three HDAC peptide substrates; end-point and kinetic experiments; real-time deacetylation rates; IC50 for HDAC inhibitors; mechanism of action.
  • Poster: Effects of Template-Directed Assembly on the Kinetics of Receptor Tyrosine Kinase Activities in Microfluidic Mobility-Shift Assays.
  • White paper - Fragment-based screening of enzyme drug targets: Microfluidic mobility shift assay improves confidence in candidate selection. Describes the use of the MSA assay as an alternative to traditional biochemical assays for fragment-based screening.

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Citations


Browse EZ Reader citations

Table of references by target, with substrate indicated.

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Other PerkinElmer enzymatic assay technologies


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