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Published On 09/20/2019 2:49 AM


Reddot Biotech Inc.

Copyright © Reddot Biotech Inc. 2018-2019 All rights reserved

Customer satisfaction is our top priority here at Reddot Biotech. We want our customers to be happy with the quality of the product, and the quality and success of the results of their experiments. To ensure that, we use high quality raw materials and are constantly working on improving the utility and functioning of our ELISA kits.

       However, we understand that sometimes, things go wrong. We are always happy to provide assistance and technical support for any issues you may have with our products. Outlined below are some common problems some users experience and how to combat them. 



Possible Causes

No Signal

After adding TMB substrate, all wells remain colourless

  • The reagents have expired
  • Adding detection reagent A, B or TMB substrate was skipped
  • Distilled water used for dilution was contaminated
  • Enzyme inhibitor (such as Sodium azide) is present
  • Wash buffer is concentrated which is not diluted in proportion.
  • The activity of the labeled enzyme is low.
  • PH value of solution is incorrect. For best results, it should maintained at 7.2-7.4

Weak Signal

After adding TMB substrate, all wells show a weak colour

  • The reagents have expired or have not been stored in the correct conditions.
  • The reagents, standard, and/or sample were not warmed to room temperature prior to conducting the experiment.
  • Incorrect amounts and/or dilutions of reagents were used.
  • One or more solutions used were contaminated.
  • The labelled enzyme is contaminated or inactive.
  • TMB substrate chromogenic time is not enough.
  • Washing procedure problems, i.e.:
    • Wash buffer too concentrated.
    • Wells were washed too many times.
    • Wells were washed too vigorously.
    • Wells were washed too long each time.

No Colour Gradient

All wells on the plate show the same weak or strong colour

  • TMB substrate was not stored in a cool dark place and protected from light exposure.
  • The incubation time was too long (which can lead to strong, nonspecific adsorption).
  • Wells were not washed adequately during the experiment.
  • Cross contamination from pipette tips or contaminated reagents.
  • Low concentration or low activation of the coated or detection antibody.

       We always recommend thoroughly reading the instruction manual before use and following the recommended procedure. Here are some more things to keep in mind for a smooth and successful experiment:

-All reagents in the kit should be stored according to the manual instructions. For ready to use kits, all components can be stored at 4oC for 12 months; for Traditional kits, The TMB Substrate, Wash Buffer (30X concentrate) and the Stop Solution should be stored at 4oC upon receipt while the others should be at -20oC.

-Before beginning the experiment, all components should be brought to room temperature.

-To ensure uniformity and accuracy of each reagent, mix all components of the kit before use.

-Keep the environment of your lab stable at room temperature (18-24oC) with constant humidity throughout the entire experiment.

-A pH between 7.2-7.4 should be maintained for the entire duration of the experiment.

-Ensure all concentrated reagents are appropriately diluted prior to beginning the experiment.

-The washing technique should be kept consistent throughout the experiment and should be done according to the instructions in the manual.

-Avoid creating bubbles in solutions to ensure uniformity of all reactions.

-Take the reading of the microtiter plate 2 minutes after adding the stop solution.

       If you are experiencing issues with a product, please feel free to contact us here for more help, or email [email protected] with the details of your experiment.

This entry was posted in Product Literature