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293T cells go wild after >50 passages??? - protein expression changed!?? (Aug/05/2004 )

Hi All,
Here is another situation regarding passage number of cells.
I've cultured 293T cells from a single vial for long time (>5months), presumbly passaged them for >30 times. The cells look absolutely healthy and normal. However, when I transfect the cells and had them express my proteins, the proteins they expressed now are no longer biologically active!! When I ran a western, I found the protein is smaller compared to protein I expressed 5 months ago using the same patch of cells. I don't think it's the problem of my construct, because I resequenced it to be correct.
Does anyone has any explanation of this?? what happened to my protein..... glycosylation, degradation.......??? What should I do next?


Hi Beautytree

There are often a problem with the cells changing after many passages. Normally, you should before you start your assays, culture as many cells as possible and cryopreserve them at the same passage number. When you make an assay you thaw cells, so that the passage number i about the same for every assay.
If you have preserved some cells at the beinning, thaw them and grow as many as possible for a couple of passages and presserve them. If you have'nt you should probably get some new cells, and start by presserving as many as possible.

Regards Carina