bands from tissue one size, from cells - size doubles - help! (Feb/03/2005 )
My protein of interest is ~30kDa - this is the size it runs at on a western blot using mouse tissue samples. However, I have been performing some WB's on cell extracts and am finding an extremely strong band approximetely at ~60kDa appearing.
From mouse tissues I see no other bands appearing other than a strong band at 30kDa, but with the cell lysates I see a faint shadow at 30kDa but a very strong band at 60kDa.
A colleague has suggested that maybe my protein has formed dimers, strongly linked that they are not dissociated in the reducing sample buffer. Could this be the case, or does anyone have any other ideas???
Thankyou in advance,
I guess its the dimer of your protein. What concentration of reducing agent you are using in your loading buffer? first of all is it mercapto ethanol or DTT? and also some times boiling the samples before loading helps. did u try that?
I use mecapto ethanol in my sample buffer, and always boil for 5 mins before loading the gel. I am trying it again today with some fresh sample buffer that I made this morning to make sure it hasnt 'gone off'.
Thanks for your reply,
Good call on the fresh buffer. If that is not it, then what manipulations are you doing to the tissue vs. the cultured cells? Is there a step that might be helping tear dimers apart in the tissue processing?
Yes, I process my tissues using trizol and a mechanical homogeniser - whereas I us a lysis buffer (tris, sds, sodium vanadate, NaF and some protease inhibitors) for my cells and quickly sonicate for 10secs.
i have been using this buffer on my cells as others in the lab are using it successfully to view phosphorylated proteins, which is one thing I want to see from my cells. I guess the trizol and homogenisation could be breaking apart any dimers.
How can I tell for sure? How do you deternmine if a protein is dimerising???