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General Arrangement Drawings – Plate+ Glass Coated Microplates – 96-1mL Manual / Product Insert

  • Version: FEB.2016
Catalog #

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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Fluorometric polymerase chain reaction (PCR) enzyme-linked immunosorbent assay for quantification of immuno-PCR products in microplates. Citations & References

  • Authors: Niemeyer CM, Adler M, Blohm D
  • Journal: Anal Biochem (1997) 246:140-145
  • PubMed ID: 9056198
Catalog #

Automated Panning and Screening Procedure on Microplates for Antibody Generation from Phage Display Libraries Citations & References

  • Authors: Turunen, L; Takkinen, K; Soederlund, H; Pulli, T
  • Journal: Journal of Biomolecular Screening 2009 3:282-293

Product Sheet: Click-iT EdU Proliferation Assay for Microplates Manual / Product Insert

  • Version: A.0
Catalog # C10499

Why does the 96-well Piko PCR Plate have a 6x16 format instead of the typical 8x12 format of microplates? Product FAQ

Answer

By using a 6x16 format, the standard multi-channel pipettors and liquid handling instruments can be used without modification.

Answer Id:: E8697

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Click-iT™ EdU Proliferation Assay for Microplates Limited Use Label Licenses

Limited Use Label Licenses
Catalog # C10499

Nunc Microplates Product Literature

What microplates are the best for the different modes of detection for a protein or enzyme activity assay? Product FAQ

Answer

Depending on the reagent, absorbance readings are best performed in glass or quartz cuvettes or multi-well microplates. Plastic cuvettes and microplates may work just as well as long as the plastic does not absorb light in the range required for the reading. For example, many types of plastic plates absorb UV light, and cannot be used in this wavelength range. Check with the manufacturer’s specifications as to the usable wavelength range for the plates you plan to use.

For fluorescence, black-walled plates are recommended to limit crosstalk between wells. Whether these plates have black or clear bottoms depends upon the optics of the instrument (i.e., Are samples excited and read from the top or excited from the top and read from the bottom?). Cell-based assays should be done in optically clear, flat bottom microplates, primarily because cells will settle to the bottom.

For luminescence (bio- or chemiluminescence), white plates are recommended.

Both fluorescence and luminescence-based assays may be performed in clear 96-well or larger well size plates, but 384-well and smaller well microplates must be either black-walled or white plastic, respectively.

Answer Id:: E15841

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Manipulation of mRNA on Microplates as a Platform for Molecular Diagnostics and Gene Discovery. Citations & References

  • Authors: Misuhashi M
  • Journal: Biotechnol Intl (1997) 1:329-329
  • PubMed ID: 0
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Chemi-luminescent Alkaline Phosphatase Detection in White Clear Bottom Microplates Product Literature

Microplates for Chromatography-Certified - General Arrangement Drawings Product Literature

Direct reverse transcription-PCR on oligo(dT)-immobilized polypropylene microplates after capturing total mRNA from crude cell lysates. Citations & References

  • Authors: Hamaguchi Y, Aso Y, Shimada H, Mitsuhashi M
  • Journal: Clin Chem (1998) 44:2256-2263
  • PubMed ID: 9799751
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