One or two bands with different protein extraction buffer - (Aug/16/2006 )

Dear all,
I dectected two bands in cell lysates with RIPA buffer (PBS, 1%NP40, 0.5% sodium deoxycolate, 0.1% SDS), while detected only one band in that treated with PBS/Triton (PBS , 1% TritonX-100, 50mM NaF, 100mM Na3VO4).

Is there any possibility that the second band that missing in PBS/Triton is pelleted with nuclei, or can we say SDS in RIPA might seperate the second band from nuclei or sth unknown in the pellet?

If so, how can i test this?

Thanks in advance!

probably the other band is an insoluble part of the protein. And that its being extracted with SDS buffer.

extract again with the PBD/triton and spin it down so that u could aliquot the supernatant and the remaining cell pellet, dissolve the pellet in SDS buffer. And run them on a gel.

Also search for literature which might describe different form of ur protein.

Just guessing !!!

triton cannot extract agregated protein so u will only have protiens which r readily soluble in the PBS/triton buffer. And SDS which would extract kind of nearly everything is y have the second band.

It also possible that with triton u could extract only a % of the total protein and the conc. of second band was probably not sufficient to b seen and with the SDS buffer, u extract completely so the second band is visible.