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In my enzyme activity assay, some values on different microplates (same type, brand, and batch) seem to show consistently high deviance, even for different assays. What could be causing this? Product FAQ

Answer

See the possible causes below.

If using an automated dispenser, there may problems with certain fluidics lines (air bubbles, blockage, etc.). 

Depending upon the optical design of the microplate reader, it may be possible that you have ‘hot spots’ were light is being obstructed or deflected, either during excitation (for fluorescence-based assays) or during emission. To determine this, you can try the following:

For any assay: Fill every well of a microplate with water, with every well filled with the same volume. Take readings from these wells. 

Using another microplate: 

  • For absorbance-based assays: Fill every well of a microplate with a reference dye (ideally of the same color as the assay dye), with every well filled with the same concentration of dye and the same volume. 
  • For fluorescence-based assays: Fill every well of a microplate with a reference dye (ideally of the same fluorescent color as the assay dye), with every well filled with the same concentration of dye and the same volume. 
  • For a bio- or chemiluminescent-based assays: Fill every well of a microplate with either substrate and purified enzyme or, substrate and chemical, with every well filled with the same concentration of substrate/enzyme or substrate/chemical and, the same volume.

Analyze the microplates, collecting the relevant data (either absorbance units, RFU or LU) from every well in the microplate. 

Calculate the average value and then search for deviations from the average. If performing the analysis on an Excel spreadsheet, it may help to color the spreadsheet cells per the deviation for more immediate visual inspection. For example, average values are shaded grey, but below average values are shaded blue and above average values are shaded in red. Compare the data with dyes or substrates/enzymes with the water plate. Water plates for luminescence detection should not provide any LU (positive or negative) or only very low numbers. Any large values may denote light leakage in the analysis chamber. 

Are you able to detect any trend regarding where on the microplate these deviations occur? That is, do they occur in a specific area of the microplate (only at the edges or in certain column or row)? Is the data consistent from one area of the microplate to another? Deviations that occur from one half of the microplate to another may be due to the plate not being completely flat. 

If using automated dispenser, you should examine more than one microplate, to determine if there is dispensing errors within certain areas of the microplate. 

Answer Id:: E15855

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  • Authors: Mann CM, Markham JL
  • Journal: J Appl Microbiol
  • PubMed ID: 9633651
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