extract proteins for WB - (Sep/18/2007 )

Hi, there,

I have a quick quesiion about harvesting proteins.

Generally I treat cell in 100mm dish by growth factor. Then I scrape the dish in 1.5mL cold PBS. After spin down, add 50-60uL lysis buffer(protein concentration is good to do western blot). By doing this, I am wondering some proteins will be degraded or some signal pathway will be activated. So I try to scrape cells in lysis buffer. However, 50-60uL lysis buffer in 100mm dish is almost nothing, how can I scrape? Does anybody have any good methods to harvest proteins?

Thanks for sharing your experience.

well i think that if your plate is on ice when scrapping and if you're using cold pbs, the proteins should remain at their state of origin.
i prefered trypsinise my cells because i needed to adjust my protocol precisely on the cell number.

Thanks for your reply.

I am wondering after trypsinazation, is there other effects on cells, such as stimulation or giving extra stress to cells?

Hi,

I am just wondering if the WB is so sensitive that you would see a difference by harvesting cells the way you described it?

Cheers

well i don't think cells are stressed that quick.
Anyway if you work on cell stress i gues they may be more appropriate techniques, which btw i don't know.
The trick is to was more cells, to remove trypsin.
Also i wash also a lot cells after scraping to remove debris and maybe proteases or rnases remaining free.

in in vitro transcription assay, results are better when trypsinisation is done