Hello,
For one of my experiments, I enriched cells by staining them with a PE-conjugated antibody and performing a positive anti-PE enrichment to obtain a population that was 64% PE+. After 4 days of subsequent culture in normal cell media at 37C, I stained the cells again with the same antibody and saw a drop to ~49% PE+. Is it possible that my marker remained blocked by antibody but that the conjugated PE either denatured or became separated from the antibodies? Or is this unlikely?
Thanks,
Ragan
Hi Ragan Pitner , I have the same question! I wondered if you had the answer already.
I use the MACS positive selection strategy to enrich for a specific population of cells, after which I culture them for 5-7 days and would like to test functional activity of the receptor used for positive selection. Does the antibody stay bound to this receptor, and is there a way to dissociate it in a way that would still allow for reculturing of the cells after?
Thank you,
Vinita
Hi Vinita,
Unfortunately I never got a satisfactory answer to this question, but I found that for my purposes extended culture was detrimental so the point became moot. One idea though - perhaps you should investigate turnover of your receptor and whether it gets internalized, because if so then it's possible you may lose the bound antibody to normal endocytosis over time.
Good luck,
Ragan
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