I've been trying to detect mScarlet protein purified from E. coli via an ELISA assay. My protein has two tags: an N-terminal Strep-tag and a C-terminal His-tag. To do the ELISA I've used Streptavidin Coated Plates (https://www.thermofisher.com/order/catalog/product/15500#/15500) so that the Strep-tag attaches to the plate and then using an anti-His antibody for the detection of the protein. However, not only we've observed low signal on our samples which are similar to our negative control.
On another pilot test I used 96 well high binding cell culture plates and no BSA blocking, and this time around the negative control shows more signal than the purified mScarlet samples.
If anyones knows what could be going wrong, your help would be very much appreciated.
The main issue is that you did not get a high/significant signal from your protein.
It is possible that the HIS tag is not exposed, could you try Western blot to see whether the protein is expressed. you could also do indirect ELISA to test both of the tag. Just coat the protein on the ELISA plate, block, and adding anti-tag antibody and conjugate.
If the protein is being made correctly, there should be no need to "detect" it. The cells and protein solutions would be bright orange, easily visible by eye. Are they? If not, then something is wrong with your process. I can't begin to guess what it might be, without seeing all the details.
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