I am working with leukemia/floating cells. Typically, I spin down the cells, wash them with ice-cold PBS, spin again and then lyse the cells immediately.
I am thinking If you could snap freeze the cells after washing with PBS, then store the samples at -80C until you collect a batch of them and make cell lysates at the same time. One concern is that would this processing affect the phosphorylation status of your proteins of interest?
I think it should not be a problem, while others hold different opinions. Please let me know your opinions. Thanks a lot.
Hi Rongqing,
I would not recommend freezing the cell pellet in case you want a subcellular fractionation (for example, cytoplasmic & nuclear extract). The reason is that during the freezing process, ice crystals are formed and those would pierce intracellular membranes.
Otherwise, if you want to get a whole cell lysate, you should be fine. I think it is a good idea to add proteases/phosphatases inhibitors to your last PBS wash.
Best of luck!
By the way, I am planning to use dry ice to do the "snap freezing".
hi Rongqing Aaron Pan
I don't see any wrong freezing up your cell until use. However, I would think carefully doing this since stress could activate many of proteins.
good luck
Your best chance is to use phosphatases (serine/threonine and tyrosine) inhibitors in your collecting medium. That way you make sure nothing is going to change in between you thaw your cells and lyse them
Yes Friend Pan,,
no worry.. you are absolutely ok,, i am also doing like this with my phospho concerning lysates..
good luck...
Washing the cells with PBS and storing the pellet at -80 C will not affect the phosphorylation status of proteins. I have done this without concern. You can also sonicate the cells in phosphate buffer (3 times, 15 sec bursts) and store the lysate at -80 C. If you are worried about having enough protein, you can combine lysates or simply grow up your cells in larger dishes/flasks.
Thanks everyone for your answers.
BTW, I add fresh phophatases/protease inhibitor to the SDS lysis buffer each time before I use it. The lysates can be kept at -80C for future WB.
I think Avoiding repeated Freeze/thaw cycles is critical to minimize possible protein degradation.
Again, Thanks for sharing your experience.
No problem in snap frozen,
Just as a suggestion and just in case you do not know..be careful with your blocking buffer, use 5% BSA instead of milk , milk has casein which is a phosphoprotein ...it could be an issue in terms of background..
Jose.
Hi Rongqing,
I would not recommend freezing the cell pellet in case you want a subcellular fractionation (for example, cytoplasmic & nuclear extract). The reason is that during the freezing process, ice crystals are formed and those would pierce intracellular membranes.
Otherwise, if you want to get a whole cell lysate, you should be fine. I think it is a good idea to add proteases/phosphatases inhibitors to your last PBS wash.
Best of luck!
Hi I always start with equal number of cells. between the controls and then I lyse the cells as soon as the treatment is over with SDS-loading dye and load equal volume of lysate. For example for a 6 well plate, I wash the adherent cells with PBS and then add 200ul of 1 X SDS-loading dye and lyse the cells, the lysate will turn viscous, we sonicate the lysate to decrease the viscosity and load 50 ul on gel to detect MAPK phosphorylations. Good luck.
- Badges
- Science method