I have been trying to get my ELISA running for a while now and at the moment don't know what else to try.
I am trying to detect Flotilin-1 in lysates of human monocytes.
Primary antibody is from cell signaling https://www.cellsignal.de/products/primary-antibodies/flotillin-1-d2v7j-xp-rabbit-mab/18634
Cells were lysed with 1% Triton. Cell lysates were put onto 96-well plate with high-protein-binding capacity (after cooking them for 5 minutes at 95°C) overnight at 4°C. After washing three times with PBST and blocking for one hour at 37°C using protein free blocking (https://www.thermofisher.com/order/catalog/product/37584#%2F37584) plate was incubated with primary antibody 1:25 in 1% BSA in PBST overnight at 4°C. After washing three times for 5 minutes with PBST secondary antibody was put onto the plates for one hour at 37°C. Secondary buffer is 3% BSA in PBST. So far I tried different secondary antibody concentrations (1:2500, 1:5000, 1:10 000). I then washed 5 times 5 minutes. TMB kit was used for detection.
The problem is, that only in the 1:2500 dilution I see any change of colour but the signal-to-noise ratio is pretty poor and I get only 1,5-fold change in my cell lysates vs background (defined as signal in wells with cell lysates treated with only secondary). In the higher dilutions I get very few signal.
As 1:2500 is quite a lot of secondary, I suspect that all the signal I see is just unspecfic binding of the secondary. My guess at the moment would be that the primary antibody is not working in ELISA, although I previously tested the antibody successfully in Western Blots.
Any help is appreciated
I would recommend testing the primary antibody on serial dilutions of flotilin standard, if those are available. Best wishes.
I would like to recommend
different dilutions of the primary antibody. The manufactrure instructions showed different dilutions for using the 1ry in different methods. They did not mention the recommended dilution for working in ELISA.
Try diff. dilutions of the primary .
I think the problem in the Ab dilusion..
It's better to try several dilusions
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