Detecting proteins in Mouse embryonic fibroblast - Any tips on making cell lysates of MEFs? (Jan/21/2006 )
I have previously been working with inducible cell lines that overexpress the protein of interest that I am working with. All of that was done to optimise the western for the detection of this protein that is normally lowly expressed. I have been using 2X sample buffer to lyse the cells.
However, I have now shifted focus to the more important bit of my project, to see how much of this protein is expressed in mouse embryonic fibroblast, which is proving a little difficult.
This cell-type has complicated characteristics:
- cells are large, one MEFs-confluent T75 flask gives me approx. 6 million cells at best. Not quite enough to collect the proteins needed to make whole cell lysates.
- this cell type has a tendency to senesce if the culture conditions proves to be uncomfortable for them.
Just wondering if anyone have any experience workin with MEFs, any tips would be greaattlyyy appreciated. THANKS!
Dont know if I can help you too much, but if a T-75 does not give you enough cell for your protein, why not subculture into a T-225?
hmm.. MEFs dont last long in culture (at least in my hands) and get senescent pretty fast. had the same problem as you - trying to get enough to check protein levels. i noticed, that i can get them growing better when i dont dilute them to much. i used to split them 1:2 to 1:3. the "younger" the split- the smaller the cells, and better growth.