I have used an antibody (polyclonal rabbit) against an specific type of my protein (an ubiquitinated variant) in a WB. I`ve seen the band, which is alright (molecular weight checked with marker and a different antibody that recognizes the wt-protein and uni-protein). It works really fine and it is detected with the Licor-odyssey system.
But when I did the dot-blot, I can no longer see a difference between my negative control and my positive control. (Negative means that no signal is expected) What I get is the same amount of signal (according to image studio lite) in the positive and in the negative controls which is really above zero.
When you run your WB, was there any non-specific band(s) or only one specific one? If your antibody has generally high background, you specific signal will be loss on the dot-blot.
I would like to ask whether there were any differences in processing the two immunoblots in both the techniques. Any number of differences could account for a different outcome in dot blot as compared with a WB.
If I understand well, you want to detect an ubiquitinated variant with a polyclonal antibody. Which antigen is it directed against? The ubiquitinated peptide? If so, it is likely that there is some non-specificity, e.g. any ubiquitinated protein can be detected by the antibody, even very faintly and the resulting background is either dispersed all along the gel lanes on WB membranes, which is OK to see your band of interest, or is concentrated on a small area with the dot blot technique, which cloaks the signal. You can try different antibody dilutions or incubation times to favor the genuine signal.
How did you process the dot plot? if you have just spotted your cell lysate on the membrane in a concentrated spot that could be the issue. Usually you wold load 30 ug of total proteins in the gel and then separate it by SDS page, once separated the the individual bands would have a concentration in the nanogram range. If you spotted 30 ug of cell lysate in a concentrated spot you may increase the noise to signal ratio.
Thank you all. I will try to answer your questions the best I can.
Dear Richard, when I run my WB there is only one specific-band and my antibody has no high background (on WB)
Dear Divaker regarding the processing as far as I'm aware the only difference is vacuum in dot-blot, and running and transfer in WB. But blocking, incubation with primary and secondary antibody, times and washing steps are all the same. I
Dear Philippe. I`ve made a mistake when describing my issue. The antibody is monoclonal and is directed against the residues of the surrounding area ubiquitinated. I have tried antibody dilutions but that didn't work.
Dear Sergio. I`ve done serial dilutions of my lysate and still see the same amount of signal in the dots.
Probably you have a no specific band in your WB or in the proteins that are too small to be seen in the WB but that are in the whole mix you use in the dot-blot. Another possibility is that the amount of material used in the dot-blot it too high and you saturate with antibodies. Try to use less primary antibody in the dot-blot.
Antigen presentation is probably different in the WB and dot blot context. That may be your problem - particularly since you're using a monoclonal directed against a small, specific region. SDS-PAGE and transfer are thoroughly denaturing your protein, and your Ab is recognizing it in that context. How is the sample treated before applying it to the dot blot (detergent, reducing agent, temperature) -- and how are those conditions different from the SDS-PAGE sample? I suspect that your mAb may not have access to the epitope in the dot-blotted samples.
You could also be experiencing some masking of epitopes on the dot blots by other proteins in the sample. Everything is concentrated into a very small area, and there could be steric crowding. That could exacerbate the antigen-presentation issue I described above.
Dear all
Thank you I solve this issue using less primary antibody and lower detergent concentration.
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