My intention is to do an ELISA with brain homogenate coated on the base of a plate -> Adding a sample that might have antibody that binds to the homogenate -> Add a secondary and look for binding
Despite finding binding in westernblot, I fail to see any signal on ELISA. It seems to me that it is being washed away and not being coated well. There is no issue when I use purified protein. I use coating buffer and NUNC non sterial ELISA plates - coat for 16-20 hrs.
I used CHAPS buffer to homogenize so that its kinder to membrane proteins (which are of primary interest). Is the detergent action preventing to bind?
Is there changes to coating that I can do? Can detergent prevent coating - but how can I homogenize without detergernt if I am looking for brain membrane proteins?
I think the detergent is likely to be the problem. ELISA plates have a hydrophobic surface to which the proteins bind. Detergent can block the hydrophobic surface.
Add 100 µL of diluted brain homogenate to each well of the ELISA plate.
- Ensure even distribution by gently tapping the plate or using a multichannel pipette.
- Cover the plate with a lid or sealing film to prevent evaporation.
Hi Vasishta,
It seems that the CHAPS detergent might be interfering with protein coating in your ELISA. You could try removing the detergent via dialysis or buffer exchange, increasing the homogenate concentration, or switching to a detergent-free coating buffer.
However, since you're working with membrane proteins, I would recommend considering immunohistochemistry instead. IHC avoids the detergent issue and allows for protein localization directly in tissue, giving you more insight into location, distribution and context.
Best
Stöpa
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