Everyday I do a western blot and I am left helpless for new things keep on surfacing. Can you please explain looking at my poncean where is my experiment going wrong to give me varied thick and thin western blot bands? I carry out western blot by quantifying it using bradford assay followed by using lysis buffer---95C boiling--- loading 30ug of protein. running on precast gels for 1hr and half at 150V---transfer for 2hrs at 100V--and I look at the poncean and I see this.. Please help me out? Western Blot is taking my sleep away and its frustrating. Thank you
Hi Rahmat,
To me, your ponseau stained blot looks fine! There is no problem in SDS-PAGE and transfer. That is the purpose of doing ponseau stain.
Amount of lysates on lane 1,4,6 and 9 appear to be more compared to the rest. But, they are equal at least. You should proceed to western blotting. Actin (eg) is your ultimate loading control. Do not overexpose actin signal (it should appear as separated band on each lane).
Even though the blots might not perfect, densitometry analysis is the accurate quantification for signal of interest.
I also think there is nothing wrong with your picture. The gel and transfer were good. The higher abundance of some of the proteins leads to more intense and broad bands. Besides some of the proteins might have different post translational modifications like glycosylation, phosphorylation and so on, which might cause the bands to be not so sharp. You can go ahead with your western blot development, everything should be fine.
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