After transfer, i stained with ponceau which revealed nice bands, however after secondary incubation, washing and adding the chemiluminescence substrate, the image produced looks like this. I am not sure what the problem could be?
The problem appears to be the imaging instrument, as i can see proper squared lanes aligning exactly adjacent (something quiet impossible), please share the ponceau image itself so a clear conclusion can be drawn
After looking at the ponceau it seems the transfer went well, yet there are two possibilities of such a blot pattern, either you protein of interest is fragmented in each sample (as multiple bands be seen) which hints the issue in protein extraction protocol (if there was a single band expected per sample) or the ChemiDoc Instrument have gone faulty with that is generating such pixelated images, have you tried stain free ?
no i have not tried stain free yet, i think i can explore that portion. also with regards to the protein, how would i be able to tell its fragmented before running the western? Because i run a protein concentration assay after the extraction before the western. Is there also a way to prevent fragmented protein?
Yes, protien fragmentation can have two reasons, either the protien have multiple fragments already in its natural state which simply separate over the SDS PAGE or your extraction buffer failed to prevent protinases activity (in that case inhibitors or a strong extraction buffer)
alright, but this is the second gel i made with the same extraction buffer, in the first gel, after imaging there were bands, so i am not sure that could be the reason. this is what the first gel looked like.
Hi Serena.,
Jatin may be right about the faulty acquisition by the equipment.
What's your secondary ab concentration? Seems to high, try using it at 1:20K-1:100K dilutions. You could also run the gel a bit more to better separate the proteins, or do you need them packed like that?
Best
Thank you Sebastián Dupraz the secondary antibody concentration is 1:1000. I would try your recommendation. The last picture was a trial. But I rerun it longer making sure the protein was prop separated.
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