ELISA Technical Resources

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Troubleshooting guides

Troubleshooting guides

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ELISA Troubleshooting: Saturated Signal

If your ELISA signal is too high, the results of the experiment can become unusable. Saturated signal can cause wells to appear uniformly reactive, or cause the standard curve to become unusable. Before you repeat your ELISA experiment, read these troubleshooting tips to identify possible sources of your saturated signal error, and solutions to solve it.

Problem Cause Solution
Substrate problems Excessive substrate Decrease substrate concentration
Incubation time too long Reduce incubation time
Reduce incubation temperature
Substrate activation before use Make substrates immediately before use
Plate error Insufficient washing Follow manufacturer’s washing instructions
Thoroughly drain plate after washes
Plate sealer not used or reused Cover plate with plate sealer during incubations
Use fresh sealer every time
Plate read at incorrect wavelength Use recommended wavelength/filter
Check that plate reader is set up for the type of substrate used
Antibody problems Excessive antibody concentration Reduce antibody concentration
Nonspecific antibody binding Use a different formulation of coating solution
Use Boster antibodies guaranteed to only react with their targets

Keywords:ELISA, Troubleshooting, Saturated Signal, O.D., Off Scale

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