Hello!

I have three questions: 2 easy ones and 1 a bit more difficult...

1) i'm currently precipitating protein with cold acetone... after i normally remove it with a pipette and I dry the pellet in a speed-vac... I read that we should avoid to dry the pellet... Why? How can i get rid of acetone? (after I need to digest the proteins with trypsin)

2) always concerning acetone precipitation: i often observe an upper phase (lighter than acetone): are they lipids? i tried to extract them with CHCL3/MetOH (9:1),... is it a good idea?

3) and now the difficult question (LOL): i would like to produce a yeast strain deficient for lysine... Do I have to delete only the Lys gene (one of them) or should I delete genes for other amino acids which are by-pass products for Lys biosynthesis? (or maybe: do you know about a website containing information about the yeast biosynthetic pathways, where can I found the answer to my question?)

Thy 3 times!

Well, I only have a suggestion for the "difficult" question

Wouldn't it be easier to get a mutant yeast strain for that gene instead of making it yourself? There are yeast mutants for several amino acids production (I duuno if Lys is one of them though).

But maybe it's cheaper to do it yourself right?

1) completely dried pellets are often very difficult to resuspend and may be more highly denatured.

1.5) you can get rid of the residual acetone by dialysis or, better, a desalting column (sephadex g-25).

2) they may be lipids or lipoproteins (or something else that is lower density than the acetone/water mix).

2.5) resuspend or extract them? why? do you need anything in there? are you losing your protein of interest and looking for it?

they may be denatured. you can skim some of it off the top and look at it on a gel. then decide if you want to see more. but it probably holds no value to you at this time.

or you could collect it with the rest of your pellet and continue with your preparation.

Thx for the answers:
I just don't understand why should i bother about the fact that dried pellets contain more highly denatured proteins, because after protein precipitation i end up in any case with denatured proteins (i don't care about their function... i just need to digest them)

Concerning the upper phase after acetone precipitation, probably i didn't explain myself correctly: i'm not interested in those "lipids"... i just want to remove them because after digesting my proteins i want to do a mass spectrometric analysis and the cleaner my digest is the better it is...

precipitation doesn't denature the protein. it just makes it less soluble in the medium that it was in (note: tca does denature).

if you expose the protein to air then you may denature the protein.

if you just want to remove the floater then skim it off or stir it up (only the top, stay away from the pellet) and pour it off with the supernate. i skim and stir and pour what i can't skim.