Hi I am trying to detecting ZO-1 (~210 kDa) in colon tissue. I used 1 hr transferring to the blot and I am pretty sure I wash the membrane very well after secondary antibody and HRP. But I still got this high background band. Although I can find my target protein.
Could you please give me any suggestion on how to make this blot with lower background?
Thank you
I'm not sure which band is your target protein, could you include your molecular weight markers in the image. A little bit of background is usually considered okay, and can often be cropped out when making figures for publication. Also, I'm not sure what you have loaded in the gel, but if you haven't already a good control would be to run an extract from the same cell type that does not include your protein (or epitope tag) of interest so that you can identify if any bands are due to non-specific antibody binding; any bands that are smaller than your protein, but do not show up in the negative control are most likely due to partial degradation of your protein, which you may be able to reduce by alterations to your extraction and preparation protocol. Unfortunately, without more detailed labelling of the standards and what is loaded in your blots it is difficult for me to say for sure what the reason is.
Hi Hollie, Thanks for the suggestion. The top one with most intensity is the target protein I am looking for it is between the protein ladder of 250 kDa and 150 kDa. I loaded 30 ug of protein extract from mice colon tissue lysated with RIPA buffer supplemented with protease inhibitors.
Hi, if the top-most band is your protein of interest then my guess is that most likely the lower bands are related to partial degradation. You can assess for non-specific binding by using an extract that does not contain your protein (or epitope tag) run in parallel as I explained previously. From what I have experienced /seen with Westerns what you are seeing is generally considered acceptable, as you can crop out the lower bands to make a cleaner final image for publication.
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