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Curved bands in Western Blot


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#1 polonese

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Posted 22 February 2013 - 07:28 AM

Hello everyone,

i`m experiencing some problems with my blots. As you can see in the picture the bands seem to frowning and are also somehow connected. First i thought that it might be because of a too high voltage (70V) during SDS-PAGE so i reduced it to 60V. I also tried to use some bigger wells to avoid those connections but nothing really changed. Sometimes it even looked worth.
So do you think that this could be the reason of a wrong salt concentration in my running buffer?

I`m using 2X Laemmli to isolate my proteins and a 12.5% bis-tris self-cast gel. The antibody i used was against H3 (17kDa).

Any help would be greatly appreciated.

example.jpg

#2 GNANA

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Posted 22 February 2013 - 01:46 PM

i suspect the pH change in the LOWER gel buffer !!! or more detergent in the lysate....
I would prefer being perfectionist rather than a passionist in Research.

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#3 mdfenko

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Posted 25 February 2013 - 07:15 AM

you are seeing diffusion through the sample wells' walls. the strange part is that the sample in the wall is migrating faster than the sample in the well. this may be due to pH differences in the stacking gel and the sample and running buffers.

you should load the wells as rapidly as safe (you want to avoid back mixing with the running buffer) and start the run immediately to limit diffusion between the wells. you also don't want sample to overflow the wells.

as for your question about salt in your running buffer, i hope you meant the components of the buffer and not actual salts. salts will cause all sorts of problems with electrophoresis.
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