Yet another post regarding diffuse bands

[prlitt]

Hello everyone,
This is my first post and I am hoping that someone can give me some insight into a problem I am having with SDS-PAGE gels. I cast the resolving gel the day before I cast the stacking gel (I overlay the resolving gel with water overnight) and all buffers are made fresh on the day of use. I have tried running the gels at different voltages, ranging from 50V to 200V, using the dye front as a guide for run times. I have also tried different acrylamide percentages and found that 6% seems to give better results for my protein of interest(HSP 70, ~68-72 kD). I am attaching a picture that shows what the majority of my membranes look like after I blot them. I have seen worse, but I would like to have less "fuzziness" between the bands. A previous student in the lab where I work had great looking, tight bands, so I know that it is attainable. Unfortunately he is no longer in the country and I cannot find any contact information for him. Thank you for any help.

[postdock]

Hello sorry for the late reply but it seems you are having gel issues. When you make your gel, make sure you use fresh reagents and mix them well (very well) while making your gels.

Also make sure you have a stacking gel well formed, and the gel is in general well formed. Also your running buffer is probably faulty along with your running current settings.

I would set it at constant current and make sure you use carefully made running buffer that is a freshly made and not reused.

You seem to be almost there as your bands are relatively sharp however it is not as straight and as sharp as they can be.