Hello everyone,
as you can see, I have troubles with my western blot assay. This is a nitrocellulose membrane dyed with Ponceau S. The lowest part of the membrane has a curvature in each sample lane and I don't understand why. Sockingly, the molecular weigh standard it's ok in this part. I'm doubting about the samples origin (the samples are from rat liver) or the current during the electrophoresis (nevertheless, this should affect the molecular standard also). The acrylamide percentage of this gel is 10% so the problematic part it's between 10 to 37 kDa
Thanks in advance for your help!.
Such effects might be caused by the presence of salts or detergents in your samples (tissue extraction buffer). To test if this is the case you can run another sample (in another buffer) side by side on the same gel.
Thanks Viktor for your answer.
The point is that this buffer (RIPA buffer) has been used by other people at the same time and they dind't have this problem. Anyway, I'll keep your recommedation in mind.
Best wishes.
Might be due to an excess of proteins in that area. In the low MW area, apart from your native proteins, you also have many fragments coming from the degradation of bigger proteins. In some staining techniques, such as ponceau or comassie, when you have a saturation in the system (too much protein) color, red in the case of Ponceau, does not appear and you get a big white spot instead of a red one, same that you have in your lower part of the gel. Try to run less amount of protein, maybe it would help.
Anecdotally, I may have seen a similar effect when my final concentration of SDS sample buffer (Laemmli sample buffer) has been too high or too low.
The final concentration in your sample when you load it should ideally be:
60 mM Tris-Cl pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol.
Here is a link for the recipe of 5x concentrated LB that we have been using:
http://www.cytographica.com/lab/solutions/5XSB.htm
Thanks David, I'll follow your advice. Maybe a little less of concrentration would work better.
It's interesting what you said Alex. I'm currently using the BioRad 2x Laemmli sample buffer and it's says that the final concentration it's:
65.8 mM Tris-HCl, pH 6.8, 2.1% SDS, 26.3% (w/v) glycerol, 0.01% bromophenol blue
They are considerably higher in some cases so I'll take in count. Thank you so much!
Hi again,
I've changed the current limit during the electrophoresis and I got this result (maximum voltage permited was 200V instead of 500V) . I think the problem was that. However, I think I could limitate it more. Which current do you recommend me? I usually run the gel at 50mA, 25mA each gel and I think I should change the maximun voltage permited.
Thanks in advance :)
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