stable expression in 293

Vadim vadimpedchenko at hotmail.com
Tue Feb 24 15:29:19 EST 2004


While trying to stably express proteins in 293 cells "magical
disappearance" is frequently occured. Vector used - pRC/CMV, all
proteins are FLAG-tagged for subsequent purification. In brief, after
selecting clones in G-418 (0.5mg/ml) and growing them in 48-well
plates I did WB. 12 positive clones were transfered to 12-wells.
Another screen by WB shows strong signal and 3 best producers
transfered to T25 or to T75 directly. After reaching confluency cells
were split into T175 for actual protein production... However, to my
great disappointment, expression level by WB is miserable, although
cells are growing fine and were confluent by the time medium collected
(G-418 has been reduced to 0.3 mg/ml - that is the only difference).
This scheme was repeated couple times with the same outcome. Am I
doing something wrong? Help, please.



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