stable expression in 293

Wolfgang Schechinger hubahopp at gmx.de
Tue Feb 24 17:50:29 EST 2004


If your insert and the construct are ok, maybe the cells don't like your
protein. You could try other cells, even 3T3 might express your CMV driven
plasmid. 

You might check a recent paper by Steve Roffler on a expressionmethod that
works like a charm:
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list
_uids=14725509&dopt=Abstract

Wo




At 12:29 24.02.2004 -0800, Vadim wrote:
>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.
>
>
Wolfgang Schechinger


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