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Have You Published Using an Enzo Product?
- Was substrate added at the correct point in the assay?
See the assay procedure provided in the instruction manual. - Were the antibody and conjugate added at the correct time?
See the assay procedure provided in the instruction manual. The antibody and conjugate provided are often color-coded for convenience and to help reduce laboratory errors. - Did all of the components belong to the specific kit being used?
When customers order more than one type of kit, they can sometimes confuse the reagents between kits. - How long was the substrate incubation?
It is possible that Stop Solution was added to the plate without allowing the full substrate incubation. - What were the conditions of the substrate incubation?
If a plate is left to incubate on a cold lab bench or under a drafty area during ambient incubations, signal values (e.g. optical density) may be lower than expected. - Were reagents brought to room temperature prior to use?
It is important to ensure that all reagents are brought to room temperature prior to use, or as mentioned in the product specific instruction manual. Usually leaving the kit out on the bench top at ambient temperature for about half an hour prior to setting up the assay will be sufficient, when the reagents can be stored at 4°C. Frozen volumes take a little more time to come to room temperature. Do not thaw frozen reagents in a water bath. If a different standard/sample diluent is used (such as culture media) this must also be warmed. - Was the plate read at the correct wavelength?
See the assay procedure provided in the instruction manual to ensure you're reading the plate at the correct wavelength. It may be necessary to check the filters in your plate reader and the program using during reading. If others are using the instrument, they may make changes to the settings for their experiment. - Were the proper volumes of reagents added?
See the assay procedure provided in the instruction manual. - What were the conditions of the incubations?
If the incubation times and temperatures are not observed, this can lead to lower than expected signal values (e.g. optical density). Pay attention that in air-conditioned rooms the temperature does not drop below 21°C. - How was the plate shaken during incubations (if required)?
If customers do not have a plate shaker, they will often use an orbital flask shaker or some other piece of equipment. This is not a problem as long as the liquid is vigorously displaced about 3/4 of the way up the sides of the wells without coming out. It is very important that the plate is secured into place. If the plate is not shaken and it is required in the procedure, a longer incubation may be necessary to bring the reagents to equilibrium. - How long after the addition of Stop Solution was the plate read?
The plate needs to be read at the correct wavelength as soon as possible after the addition of the Stop Solution. We generally recommend that the plate be read within 10 minutes.