1. When performing ELISA, we have 2 different panels: standard and sample. In western blot, we do BSA assay at first to quantify protein concentration, so if the protein concentration is too much, we can dilute the sample and redo the BSA assay. However, for ELISA, we can only know until the last step, so if the samples are way too blue compared to standard, which meant the OD of sample is higher than standard, we need to redo the ELISA again? It will be wasted of reagent and also we don't have many samples to perform again. Is there any way we can know sample is too condensed so we can dilute the sample before the experiment?
2. I made a mistake then incubate standard and sample with Avidin-HRP for too long (protocol is 30min, but I did 1hour) so my samples were much blue than standard. Do I need to dilute the sample or this effect is because I incubated Avidin too long? My friend said if the reason for this is because of Avidin so standard should be the same blue as a sample, but in this case, it was not. So the reason might be due to the condensed samples. What do you think?
For westerns, the BCA assay is needed because the gel can be overloaded with total protein.
For ELISA, this problem doesn't technically exist, and no there is no way to know if your samples will fall within the standard curve of the assay. For completely unknown samples, you have to test several dilutions. Once you have an idea, you can scale down the dilutions for future similar samples.
Now... as for leaving in the Stept-avidin for too long... You have a wash step after that anyway, and if you did your blocking correctly, it should not have caused too much of a rise in background. And your friend is right, if your standards are fine, then the analyte in your sample is too concentrated. You have to dilute your sample.
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