2 replies to this topic

[In_search_of_truth]

Hi all,

For low abundance proteins, is it a good idea to add SDS PAGE loading buffer directly onto the cell pellet? I normally dissolve my cell pellet in 100ul of PBS after spinning and then freeze them at -20'C until I'm ready to do my SDS-PAGE.

Also I'm not sure if freezing the cell pellet on its own is better or with the SDS-PAGE loading buffer if I don't do the electrophoresis straight away.

Any suggestions as to which approach is better?

Thanks

[bluedoozer]

In_search_of_truth, on Jun 19 2010, 02:35 PM, said:

Hi all,

For low abundance proteins, is it a good idea to add SDS PAGE loading buffer directly onto the cell pellet? I normally dissolve my cell pellet in 100ul of PBS after spinning and then freeze them at -20'C until I'm ready to do my SDS-PAGE.

Also I'm not sure if freezing the cell pellet on its own is better or with the SDS-PAGE loading buffer if I don't do the electrophoresis straight away.

Any suggestions as to which approach is better?

Thanks

Well, first you should not freeze the samples in pure PBS, you should add at least some protease inhibitors. Better it is, lyse the cells in RIPA buffer or TX100 Lysis buffer....
It´s not a problem to freeze the cell-pelltet in SDS Lysis buffer. We are permanently preparing our western blots in advance and after boiling we freeze our ready to load samples until the next day when we want to run the gel.

Be careful, after adding SDS lysis buffer (depending on the recepie) you cannot make a protein conc. determination any more.

I hope this infos help

[laurequillo]

In_search_of_truth, on Jun 20 2010, 12:35 AM, said:

Hi all,

For low abundance proteins, is it a good idea to add SDS PAGE loading buffer directly onto the cell pellet? I normally dissolve my cell pellet in 100ul of PBS after spinning and then freeze them at -20'C until I'm ready to do my SDS-PAGE.

Also I'm not sure if freezing the cell pellet on its own is better or with the SDS-PAGE loading buffer if I don't do the electrophoresis straight away.

Any suggestions as to which approach is better?

Thanks

You can freeze the pellets at -80 (without PBS or SDS), or you can add 1x SDS, boil it for 2 min, sonicate, and boil again for 5 min and then load the gels or freeze the samples.
Just be careful if you want to check some modifications (phosphorylation, acetylation, sumoylation...) Because for some of them is better just to do the lysis and load the gel in the same day