Sediments/debris/pellet in protein lysate? - (Mar/14/2013 )

My total protein was obtained using a very standard protocol of lysing adherent cells, sonicating samples to shear DNA followed by centrifuging them for 15 min to separate DNA/debris from protein. Total protein samples were stored at -20C for approximately 1.5 months before I thawed it out to run my SDS-PAGE and ultimately westerns.

I saw that there is some kind of foreign material in my lysate in about 80% of samples I'm going to run. It looks like DNA residue or some kind of crystallised debris. It doesn't resuspend into the protein lysate but remain as pieces of debris. Anyone had this before? Could it be some kind of contaminate (fungi or yeast)? or Leftover DNA which was not cleared from the spin? It's most likely not DNA since it DNA pellets were well separated from protein suspension after lysis.

What's your lysis buffer?

Could it be that your protein concentration is high and some of it has come out of solution and precipitated? This are usually hard to get back into solution.

Just a thought

almost a doctor on Fri Mar 15 09:49:41 2013 said:

I'm using a lysis buffer for phospho-proteins with the following:
Tris
EDTA
NaCl
Triton-X
NaF
Na deoxycholate
Na2VO3
Protease inhibitor cocktail

This is the first time seeing observable amounts of sediments in my samples. Could it be that I've stored my sample for too long at -20C?

almost a doctor on Fri Mar 15 09:49:41 2013 said:

I agree with this. The protein likely has come out of solution and precipitated. I observe this with some of my proteins as well after storage. I chalk it up to "it is what it is" and don't worry about optimizing buffer conditions since the buffer has maximized enzyme activity.

The only thing I do is centrifuge the sample and pipet off the top and leave the precipitated protein behind. You can try to resuspend the precipitate, but I have yet to accomplish that (most methods will destroy the active proteins).