Hi everyone, I have been running into this issue for quite some time. I notice that the intensity of bands that are towards the middle of my blots are always higher compared to the edges. (The first and last well of the gel) Is there a reason for this? Along with that, the samples that are ran on the edge always tend to be slanted as well. I am not sure if this issue is caused by insufficient transfer of some sort as well as during the separation. I always double check with Ponceau S. after transfer, and quantify the lanes as well. Overall the lane intensity is fairly close to each other.
There seems to be two of the following issues to look into;
1. Keep the size of nitrocellulose (NC) membrane and the supporting filter paper same as that of the gel. If the size of NC and or the filter paper is larger, the flow of electric potential will not be uniform!
2. The anodic wire may be over stretched or lose, which is having a lesser chance.
I also think that both are results of nonuniform electric flow in your transfer apperature.
If you are using semi-dry method for blot , try wet transfer. I have observed such problems while using semi dry apparatuses for Western.
Regards
Shamsher S. Kanwar never would have thought of that! I am giving it a try today. The filter paper that we buy come in bulk, so we typically cut them ourselves. It would be great if this was an issue and would be an easy fix! Thank you.
Dhananjayan Dhanasooraj I am actually doing a wet transfer!
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