Hi, I have ran a few gels in order to get a nice Beta actin bands on the blot but have repeatedly failed (did not get a result/nice bands). The samples that I am running are rat lenses. In order to troubleshoot this problem, I ran another gel and stained the gel with coomassie (no transfer done for this gel) and found that there was no/very minute protein above the 40 kDa area.
Why is this happening? How is it possible that there's no high molecular weight proteins on the gel? Is it possible that there is no high molecular weight proteins in my samples?
By the way the amount of protein loaded is 25ug and I am not sure if I can load more than that because at 25ug, I am loading 20 ul of samples (if i double the concentration I will have to load 40 ul and that won't fit the wells).
Please help
Dear Elena,
Probably you are having problems with your transfer system. Could you describe it for us?
the larger proteins transfer more slowly in general I agree with above can you run an IgG (~150 KDa with no reducing agent) in another lane and see if it transfers?
Dear Bruna, I did not do any transfer with this gel. These are the bands that I get when I stained the gel with coomassie blue right after gel electrophoresis. Therefore, it has nothing to do with the transfer system. My biggest concern here is, is it possible that there is no high molecular weight proteins in my samples? Is it something that I should be worried about? I did not use any reducing agent to dissolve/extract my protein (no DTT or mercaptoethanol etc) - because my BCA kit is not reducing agent compatible.
Hi Bruna,
include some controls! As suggested by Amarendra definitely a MW-marker, and maybe a lysate from a different origin, e.g. HeLa cells. Maybe your samples still look the same and then, you have to conclude that rat lens samples mainly consist of low MW proteins?
Hi Elena,
It could also be that the high molecular protein are present in your lysate but they aren't entering in the separating gel. Next time try to stain also the stacking gel. If this is the problem you'll have to use reducing agents, adding them right before loading. By the way, which is the concentration of the gels you're using?
Hi Amarendra, the molecular weight marker ran and transferred well. This what puzzles me, because i initially thought that my high molecular weight proteins were transferring (if so then why was the ladder transferred)?
Proteins in the ladder are already reduced/denaturalized and in a much more pure ratio than proteins in a lysate. They are good to estimate protein size but the ladder isn't totally reliable to estimate transfer effectiveness itself.
Hi, Elena,
I have asked for the marker as I could not see it on the gel. As you have said that the marker transferred well, I would just try running a few samples under reducing conditions, as a control, and check if the bands are detectable after coomassie staining.
I ran another gel again today, this time I left the stacking gel intact while incubating with commassie blue.
Anyway I will try to find other endogenous controls that fall between the range of 10-30 kDa (in size).
From the above gel, I see there are bands with high molecular weight. You can still try transferring on to a membrane and probe with antibodies. This might give you additional information.
Seems you are having a problem during the transfer. Are you sure all the gel is in contact to the membrane? Maybe your sponges or the cassete is not aplying the same pressure to all the surface. Replace sponges and change cassete. Maybe there is the problem. Good luck!
what percent of gel u r running for beta actin?? transfer the proteins from the gel to membrane for some more time..
Hi all, turns out that the high molecular proteins in my samples have degraded hence, tones of low molecular weight proteins in gel. Anyway case closed.
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