I have had poor results with a regular ELISA so I plan to use a cell line which highly expresses my target protein as my 'coating antigen' to see if I get better results. I have never attempted this before and wonder if there is a basic protocol I could use as my starting point. I've looked online, but it seems quite variable, with a lot of suppliers of kits giving vague methods without much detail.
This is my crude method outline unless I get suggestions to do something else!
- I plan to set up the assay with a variety of cell densities as this is an untried assay, probably between 10K-50K cells per well. Overnight incubation @ 37C
- Fix cells (is this the right time to fix, my protein is a transmembrane protein?)
- Wash x 3 with PBS (gently by hand and tipping out, no platewasher)
- Blocking for 1 hr @ RT - 1% BSA was my thinking
- Repeat PBS washes
- Adding my serial dilution of protein which binds my target @ RT for 2 hrs (this, I am unsure about)
- Repeat PBS washes
- Add biotinylated antibody @ RT for 1hr
- Repeat PBS washes
- Add SA-HRP @ RT for 1 hr
- Add substrate (TMB)
Thank you in advance for any suggestions and help.
Hi Helen Reynolds,
You could consider this other method. Seed the cells in a tissue culture plate and after the culture period, wash the cells thrice with PBS. Then lyse the cells inside the tissue culture plate with a good lysis buffer supplemented with protease inhibitor. Split the whole cell lysate from a particular well into two wells of a high binding plate. One will be used to detect your target membrane protein while the other will serve as a normalizer for detecting an HKG. Proceed to block with blocking buffer, primary and secondary antibodies incubation and TMB substrate for detection.
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