Protein extraction-what's in the pellet? - (Aug/22/2006 )
Does anyone have any idea about the pellet contents in cell protein extraction?
I mean using traditional protein extraction buffter like RIPA, centrifuge cells and get supernatant and pellet seperated.
Can I see there's mainly nuclei in the pellet?
Your usual, I think. Nuclei, DNA, lipids, inclusion bodies, other cell junk, crud from your inoculating loop or scraper, dust what got in your buffer etc.
My problem is when i run supernatant and pellet separately, using the same antibody, i ditected one band in each preparation but with different MW. Does that mean the band showed up in pellet binds with nuclei or DNA, or is there any possibility that the band is a product of proteolysis. I mean is it possible that proteolysis can happen in such a short time?
In addtion, how to prove that the protein in the pellet was not or was a proteolysis product?
Well, not knowing more about your results or methods, I'd guess (and this is a guess) that your protein might be a little less than soluble and is precipitating on you in the pellet--is it a protein native to your cells or something you've introduced? If not, I suppose it's possible that there are two forms of your portein, one modified/one not, though you didn't say how different the MW was . . . and when you say "run separately" you mean separate in the wells of one gel, not on different gels? Comparing the results of samples ran and blotted on two separate occasions can be troublesome. And yes, proteolysis could be a problem if you don't have PMSF and protease inhibitors in you extraction buffer (of course, I'm not sure what cells you're working with either).
I have no idea how to prove if its a protolysis product--I've never had to do that, if you can . . . but if it's much shorter and your westerns back it up? I'd say that wasn't out of t6her realm of possibility . . .
Yes i ran them one the same gel, and the MW difference is about 10kDa.
I also suspect that there are two forms of this protein.
Any more ideas?
I guess you put SB on the pellet and boil....?
Well, maybe after that a new epitope is exposed (that you wouldn't see in the Sup because it was insoluble in the pellet...), and your antibody cross-react with it... I don't see any difference between that and geting unspecific bands on the blot.
A difference of 10kDa is quite big for any modification (though, there are proteins reported to have a ladder like pattern on the gel...). If you really think that it is a proteolysis product you can do mass spec...
Try see against what epitpe is your antibody is for and BLAST search it, maybe you'll find a nuclear protein that has similar a"a seq.
And of course, it couls be isoform... (but first exclude cross-reactivity!)