My Western blots are not running proper. I lyse cells from my cell line in RIPA buffer and boil them with Sample reagent dye and NUPage reducing agent at final conc 1x for 10min at 95C. However, when I load and run my samples on the 4-20% Tris-Glycine gel, samples in some of my lanes just seem to be partially "stuck" at the bottom of the lanes and they take much longer to start running out of the wells. I have tried to use different % gels, as well as concentrating reducing agent, but still had the same issue (See image attached). Any ideas what could it be and how to get rid of that?
Thanks so much in advance!
A stupid suggestion, but by chance can help... once I did not remove the protecting band at the bottom of the gel and it appeared a similar picture.
Furthermore, do you centrifugate the lysates to get rid of the cell debris or alternatively sonicate the lysates (ask because it did not stay in the mentioned protocol and might be a good reason)? Which running buffer do you use?
Good luck!
Thank you for your responses!!
I did transfer the gels to the membrane and used them for WB but often times I see the smear, or the lanes run terribly. The lanes that go through the gel normally run wider and normal, but the ones that don't run at the same time (as in the images) have probably less protein and they get pushed and squeezed and are all smeared and often I cannot detect my protein of interest. It is very hard to use those membranes to draw any conclusions from those transfers...
I have definitely removed the white band- one time I didn't in the past nothing was even moving, so I'm sure I always remove those since then. But thank you, that would have been an easy fix!!
I haven't tried to centrifuge the lysates after I boil them (that is something I am going to try, just in case there is some leftover debris still in there, and then load only the top of the sup on the gel). I do centrifuge lysates at 15,000rcf for 5min after they have been sitting in RIPA for 15min on ice, and I do get a nice big pellet that I don't collect, but rather transfer the sup only without touching the pellet. I pipette a lot each sample during RIPA lysis, since I unfortunately don't have the sonicator, but I have a feeling that sonicator might fix this issue, so I'm looking into getting one soon!
I use Tris-Glycine for the running gels since I use Tris-Glycine gels, I have tried 4-20%, 4-12% and 10% gels and still the same end result.
Thank you tremendously for your suggestions!!!
- Badges
- Science method