Tissue vs cell line lysate - Improve sample quality?
Posted 10 May 2011 - 07:29 PM
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?
Posted 11 May 2011 - 06:40 AM
You could also try a stacking gel if you aren't already.
Edited by proteaMatt, 11 May 2011 - 06:41 AM.
Posted 11 May 2011 - 10:52 AM
Posted 11 May 2011 - 03:43 PM
i just don't understand why there is such a big difference between the cell line and the tissue...
Posted 11 May 2011 - 05:07 PM
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).