Things to Note Before Beginning Experiment
  • All reagents in the kit should be stored according to the manual instructions.
  • Before beginning the experiment, all components should be brought to room temperature.
  • To ensure the uniformity and accuracy of each reagent, mix all components before use.
  • Keep the temperature stable at room temperature throughout the entire experiment.
  • The pH should be maintained between 7.2-7.4 for the entire duration of the experiment.
  • Ensure all concentrated reagents are appropriately diluted prior to beginning the experiment.
  • Washing technique should be kept consistent throughout the experiment and 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.
Poor Standard Curve

Possible reasons:

  • Degradation of the standard due to time or improper storage
  • Standard curve dilutions were prepared incorrectly
  • Pipetting errors leading to inconsistent concentrations
  • Standard curve data was plotted improperly or without using appropriate software
No Signal

Possible reasons:

  • The reagents have expired - check packaging for expiration date
  • Adding detection reagent A, B or TMB substrate was skipped
  • The distilled water used for dilution was contaminated
  • An enzyme inhibitor (such as Sodium azide) is present
  • Wash buffer is too concentrated and has not been appropriately diluted
  • The labelled enzyme is contaminated or inactive
  • pH value of solution is too high or low. All solutions should be maintained at pH 7.2-7.4 for the entire experiment
Weak Signal

Possible reasons:

  • The reagents have expired or have not been stored in the correct conditions - check packaging for expiry date
  • 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 was not incubated long 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

Possible reasons:

  • TMB substrate was stored incorrectly (not in a cool dark place and protected from light exposure)
  • The incubation time was too long (can lead to strong, nonspecific adsorption)
  • Wells were not washed adequately at one or more steps
  • Cross contamination from pipette tips or other contaminated reagents
  • Low concentration or low activation of the coated or detection antibody
Poor Replicate Data

Possible Reasons:

  • Wells were washed inconsistently or insufficiently at one or more steps 
  • Reagents were mixed or prepared inconsistently 
  • One or more solutions were contaminated
  • Bubbles formed in wells

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