I am doing western blot and after development I can see that one/ two lanes have no band. Perhaps, next day I am doing same gel run with same sample with no change in protocol, this time another lane (I mean another sample) shows no band though the previously problematic bands appear this time. This also happens that after stripping when I am reprobing, sample with previously no band shows band this time. What can be possible reasons behind this paradoxical appearance of bands?
someone please help me!!!!!!!!!!!!!!!!
Hi,
what kind of buffer are you using for your sample?
if it leaves your protein sample sticky/slimy and hard to pipet it might be you are not loading any protein on your gels at all.
I would recommend a) warming the probe pre leading and b) vortexing for 10 sec to get the protein content evenly distributed throughout the sample. Diluting the sample also might do the trick.
Please check beforhand for the requirements of your experiment and if your protein of choice is heat sensitive (in that case have a look on protein buffers for heat sensitive protein assays.
Good Luck,
Andrea
Andrea,
Thank you for your comments.
I use incomplete RIPA lysis buffer (without SDS) with 1X Protease Inhibitor cocktail for cell lysis and the proteins remain in this buffer after extraction.
We do boil samples and give a quick spin before loading and the dye added protein samples remain homogeneous and clear (not slimy or sticky). We have not tried diluted samples. I load 30ug of protein.
Can you explain what are meaning by diluting samples? should I decrease the amount of loading further?
Hi Swarnili,
true, if you are using a non SDS buffer, samples usually don't have a problem with viscosity which might effect the distribution of protein throughout the sample.
With diluting I meant do add more buffer to the sample (if it was to viscous, which apparently is not the case, so you don't need that).
some other things you might check:
Was the sample showing consistant results whilst measuring the protein( if not, it might be again a problem of the sample preparation).
Does your loading control look even throughout the gel? (again might hint to sample or pipetting problems)
Do collegues experience the similar problems (in which case it might be some problem with the equipment).
Fingers crossed,
Andrea
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