Hello everyone, I hope you're doing well. Have you ever encountered a situation like this? Recently, I added 30ug/well protein sample and ran the 7.5% gel with 90v 30min (stacking) 130v 70min (resolving gel). After transferring with nc 0.2um membrane, wet transfer 90v 60min, I did the ponceau staining. Sadly, the staining showed nothing with my sample. Although the ladder looks nice on the membrane, I'm pretty sure that the transfer is working. I've never faced this before, and I'm puzzled.
I noticed some fade smears on the top of each lane, but no bands below. Does it mean that I have no protein on each well? But I checked the samples with BCA assay, and the concentration was good. Or could it be that the proteins degraded, and I can still get the concentration? Additionally, I would like to share that these are human lung samples. When we first extracted protein and ran the western blot, it got smears on every lane and did not show any bands. Does it mean that the protein degraded in the beginning?
I'm really concerned and wondering what could have happened to my samples. If anyone has any insights, it's my pleasure to hear your ideas.
Hmm, the fact that the ladder showed up means the stain and the transfer worked. Seems likely that your target proteins didn't enter the gel. Maybe a new batch of loading buffer would help, or a longer boil time. Can you quantify the protein content of the samples via a Bradford assay or similar to make sure you have enough protein?
I did a semidry transfer today. Other conditions are the same. This is the result.
Dear Lira,
Can you elaborate the transfer parameters you run? Although you ruled out the possibility of insufficient blotting, I still believe that the problem is there. This is because it is not uncommon to face such a problem in semi-dry blotting
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