High absorbance values for samples and/or positive control

The concentration of samples or positive control is too high and out of range for the sensitivity of the assay.

Low sensitivity

Target protein not expressed in sample used/ Low level of target protein expression in sample used Check the expression profile of the target protein to ensure it will be expressed in your samples. If there is low level of target protein expression, increase the amount of sample used, or you may need to change to a more sensitive assay. Reagents not fresh or not at the correct pH Ensure reagents have been prepared correctly and are in date

Wells are insufficiently washed Wash wells as per protocol recommendations Contaminated wash buffer Make fresh wash buffer Conjugate to strong or left on too long Check dilution of conjugate, use it at the recommended dilution. Stop the reaction using stop buffer as soon as the plate has developed enough for absorbance readings. Substrate solution or stop solution is not fresh Use fresh substrate solution. Substrate should be clear (if it has gone blue, this is a sign of contamination and it should be replaced). Reaction not stopped Color will keep developing if the substrate reaction is not stopped. Plate left too long before reading on the plate reader Color will keep developing (though at a slower rate if stop solution has been added). Contaminants from laboratory glassware Ensure reagents are fresh and prepared in clean glassware. Sterilize glassware beforehand if possible. Substrate incubation carried out in the light Substrate incubation should be carri

Target protein not expressed in sample used/ Low level of target protein expression in sample used Check the expression profile of the target protein to ensure it will be expressed in your samples. If there is low level of target protein expression, increase the amount of sample used, or you may need to change to a more sensitive assay. Ensure you are using a positive control within the detection range of the assay. Insufficient antibody Check the recommended amount of antibody is being used. The concentration of antibody may require increasing for optimization of results. Substrate solutions not fresh or combined incorrectly Prepare the substrate solutions immediately before use. Ensure the stock solutions are in date and have been stored correctly, and are being used at the correct concentration. Ensure the reagents are used as directed at the correct concentration. Reagents not fresh or not at the correct pH Ensure reagents have been prepared correctly and are in date.

Confirm dilutions made correctly Standard improperly reconstituted Briefly spin vial before opening; thoroughly resuspend powder Standard degraded Store sample as recommended Curve doesn't fit scale Try plotting using different scale