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average total protein content per 293t cell


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#1 colibriv

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Posted 04 August 2010 - 02:18 PM

Hi,

well i know protein content varies according to the type of mammalian cell and it's size off course...but i wanted to have an idea of more or less the total protein content present, for instance, in a 10 cm dish of transfected 293T cells (assuming the expressed protein is not going to significantly increase the amount of protein, compairing to a non tarsnfected cell). Basically, i never worked with mammalian cell lines and i need to express a tagged protein in 293t cells and have approximately 1 to 5mg of total protein (which i will latter on affinity purify). Some literature mention they start from 20 transfecetd 10cm dishes with 20% confluent cells (i don't know why they tarsnfect 20% confluent cells). I just don't want to waste reagents and time... (i know i can measure protein content in my lysate (from one 10cm dish) with BCA kit or other kits that are detergent compatible, but at the moment we don't have it in the lab, so i just wanted to have an estimation, if possible.

thank you so much, and sorry for the naive questions...

#2 sera_tonin

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Posted 05 August 2010 - 12:09 PM

i just collected whole cell lysate from transiently-transfected HEK293 cells (not the 293T) in 10cm plates yesterday, measured total protein via BCA assay, and got an average of about 4mg/ml protein (i used 300ul PBS to collect the cells) per plate, which is about the same as others in my lab get. the cells were about 80% confluent when i did the transfection, and i waited 48 hours to collect - hope that helps :)

#3 colibriv

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Posted 05 August 2010 - 12:43 PM

i just collected whole cell lysate from transiently-transfected HEK293 cells (not the 293T) in 10cm plates yesterday, measured total protein via BCA assay, and got an average of about 4mg/ml protein (i used 300ul PBS to collect the cells) per plate, which is about the same as others in my lab get. the cells were about 80% confluent when i did the transfection, and i waited 48 hours to collect - hope that helps :)



Thank you very much!
best,




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