5 replies to this topic

[KatieF]

Hello,
I want to run a western blot with 100ug of protein from the cells (T47D).  Can I grow my cells in a 6 well plate and get that amount of protein? Or would it be better to grow them in a flask/plate? I am asking them based on the amount of equipment available to me, since I would have to order flasks, but have 6 well plates available to me.
Thank you!

[labrat612]

Are you transiently transfecting your cells with your protein of interest?

[KatieF]

labrat612, on 03 September 2010 - 06:07 AM, said:

Are you transiently transfecting your cells with your protein of interest?

No transient transfection, this cell line is already making the protein of interest (a previous study was done to show that the mRNA was upregulated, and now I am going to show that the protein is being made via a western blot).  I simply want to grow the cells and get about 100ug of protein to load on the gel ( it is very possible that my signal will be weak, hence why I want such a high concentration of protein).
Thank you!
Katie

[labrat612]

oh ok.

For western blots I've always grown cells in flasks or 10cm2 plates. Even for cells already expressing my protein of interest.
But I guess it's possible to do the same with a few 6-well plates and achieve the same results.

[tea-test]

labrat612, on 03 September 2010 - 06:20 AM, said:

oh ok.

For western blots I've always grown cells in flasks or 10cm2 plates. Even for cells already expressing my protein of interest.
But I guess it's possible to do the same with a few 6-well plates and achieve the same results.

one well of a 6 well plate has approx. 10cm²

[MJD]

It depends on how confluent the wells are when you harvest the cells.  One well should yield between 50-100ug protein.