In my last experiment was happened such a puzzling thing: I performed an electrophoresis (using a 4-20% TGS PAA gel) on samples containing Both a ~30 bp DNA duplex and a recombinant protein expected to bind.
Once I transferred the gel on a PVDF membrane and done a western blot against the specific protein, i saw the expected black bands of my protein in the region of low molecular weights, but in the region of high molecular weights, in correspondence of the lanes where the 30 bp dNA duplex was loaded, there were 3 bands negatively colored: they were white, while the protein bands, as expected were black.
Afterwards I stained with Coomassie stain my membrane and as expected my protein was not stained, but that three bands that appeared white after the western blot, yes: they were of a fantastic blue.
What were that three negatively coloured three bands in the western-blot step? Is it possible that the three bands were DNA and coomassie staining was able to stain them? Were they a putative DNA complex with my protein that having modified is charge interacted with Coomassie? how can I explain these facts?
White bands are highly over saturated detection...too much loaded/antibody to high.
I generally do a loading series/antibody dilution series to dial everything in.
White bands are highly over saturated detection...too much loaded/antibody to high.
I generally do a loading series/antibody dilution series to dial everything in.
ok, but in this membrane, as I've written, I have both black bands in the region of low molecular weights (as I expected), but in the lane where I added the DNA I have both the black bands in the low molecular weights region, and I see one and in each lane in the high molecular weights region that are white! what does it means? in each lane I've loaded the SAME quantity of protein, so what would it means a highly saturated detection???
Check this out...
http://www.bio-rad.com/en-us/applications-technologies/western-blot-doctor-mdash-signal-saturation-issues
I don't think they are non-specific bands, in fact they are also revealed by a coomassie staining. anyway thank you
Ha...I got some too today! I ran fibronectin on the pellet fraction. I need to do a serial dilution for loading and antibody concentration
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