Tissue vs cell line lysate - Improve sample quality? - (May/10/2011 )

I'm running western blots on cell line lysates and primary tissue lystes.

I get a really clear nice band for my protein of interest for the cell line lysate. the tissue one however gives me a huge smear, and the band is really difficult to make out.

(i use different sample buffers, a Tris/Tx100 for the cell lines, RIPA for spleen and a special Tris/TX100/NP40 for pancreas; I sonicate the samples, and spin down the debris, transfer the supernatant to a new tube and add 5x sample buffer, and cook them for 5 min)

Any suggestions why this may be happening and how i could improve the quality?

thanks heaps
Elissa

If you stain the gel before doing western blot does your band look like a smear? It could be possible that you need to desalt or delipidate your sample. I assume your sample buffer contains some sort of denaturing agent (DTT, βME, etc.)

You could also try a stacking gel if you aren't already.

Do you use a protease inhibitors cocktail? Tissue lysates usually have a lot of proteases that may degradate your proteins...

I do use a protease inhibitor cocktail, yes.

i just don't understand why there is such a big difference between the cell line and the tissue...

Pancreas and spleen contain a LOT of proteases, you will need to add a more (quantity) of inhibitors and include those for the more rare proteases, which are generally not found in the cocktails.

It would also be best if you can extract the tissue within 15-30 minutes of killing the animal. You could snap freeze in LN2 to extend this time a bit, but storage at -80 still results in degradation (in my significant other's experience).