Hi, I am running SDS PAGE on a few cell lysates (primary and normal cell lines) and I've noticed that there is indistinct banding on my lanes below 200 kDa, but rather sharp bands towards the top of the gel. I wanted to know if this is normal, or if there was a problem in how the samples ran through the gel.
I am using 4-12% BisTris precast gels with MES buffer for 45 minutes at 180V. Samples are 30ug in LDS sample buffer, reduced after boiling at 85c with DTT. All my buffers, including sample buffer, reducing agent, antioxidant, and running buffer were prepared fresh and bought commercially.
I've attached an image of the gel before and after transfer using the iBlot2 at P0 (dry transfer, 7 minute), and an image of the PVDF blot with poinceau
Any advice / insight is welcome!
Bands appear a little weak, either use more protein or a more sensitiv staining procedure like RuBPS
@ARTICLE{Lam-04,
title = {Improved Ruthenium(II) tris (bathophenantroline disulfonate) staining and destaining protocol for a
better signal-to-background ratio and improved baseline resolution},
author = {A. Lamanda and A. Zahn and D. Roder and H. Langen},
journal = {Proteomics},
year = {2004},
pages = {599-608},
volume = {4},
doi = {10.1002/pmic.200300587},
language = {eng},
}
At least with your MW markers I do not notice any unusual band-spreading
@Engelbert
Protein concentrations were measured by microBCA assay, and stained for 10 minutes with coumassie / poinceau, then washed in DI water overnight / for 10 minutes (gels / blot respectively)
While more sensitive staining may work, this should be more than enough to detect what I am looking for, and I am more concerned about the pattern of banding.
Hi Samir, glycosylated proteins always appear somewhat fuzzy on SDS-PAGE.
But since you are doing a blot, I presume you are going to probe it with specific antibodies. If so, then protein transfer efficiency and Ponceau S staining are not indicators of anything. What you get out of the specific antibody staining is the only thing you should be focusing on.
- Badges
- Science method