I´m running total protein in a 12.5% TRIS-Glycine gel. It seems that the samples are dense enough as they sink to the bottom of the wells. However, when I start the run, some of the sample appears to sink to the bottom of the stacking gel as if was sliding between the glass and the gel forming a homogeneous band from all the lanes. Does anyone know why this happens?
Hello Suktika Chandra and Juan Luis Gonzalez-Giron ,
It happened to me rarely, but 'often' if not attentive to one's ways of conducting.
Try not to
Hope this can resolve the problem you came across.
Are these pre-cast gels, or do you make them yourself? I've found that sometimes if the glass plates are dirty, the sample can leak out between the gel and the plate, although the blot usually looks fine if a bit of leakage occurs. Try cleaning the glass plates with 70% ethanol and Kim Wipes thoroughly before pouring your gels.
Thank you for your response Andrea. I´m doing my own gels. I usually clean the glasses thoroughly with 70% ethanol as you recommend, however from some time ago this leaking just started to happen and I don´t know why.
Did you load sample in all pockets in the gel shown in the picture? What do you see when you stain the gels? Did the whole sample spread including proteins or just the dye? Even with precast gels the dye can form a uniform line in the gel but this doesn't affect the separation. Did you make sure you removed all buffer/liquid (after polymerization of the resolution gel) before pouring the stacking gel?
Based on the appearance of the samples on your picture # 1 not all the samples move as you described. Is that possible that these particular samples have higher concentrations of DNA, polysaccharides, or just total protein, than the other samples. These observations often can be detected when any of the above contaminants present at higher concentration.
Thank you for your responses. After changing different parameters, I figured out that just by degassing the acrylamide the front line didn´t form. Actually, I didn´t think it was an important step until now.
What does degassing the acrylamide mean? I'm having the same problem...
Juan Luis Gonzalez-Giron
I am facing the a similar problem since my last two-three attempts at making gels. This is weird as I had been making gels for quite some time, and this problem is quite recent. Could you please clarify how to get rid of this?
Thanks.
Hello Suktika Chandra and Juan Luis Gonzalez-Giron ,
It happened to me rarely, but 'often' if not attentive to one's ways of conducting.
Try not to
Hope this can resolve the problem you came across.
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