Hi Fellow Colleagues:
Lately I ve been having a problem with electrophoresis
My bands across the gel run uneven: some are normal, others are narrow or wide.
All solutions are doublechecked, everything looks good
Somedays, the samples run with a problem, other days, exact same samples, in exact the same order run normal and good
I ve been trying to pin dopwn the problem. Same problem persists at different voltages. (I use TGX gels from Biorad. Run at 100V for 100 min; or at 200V for 44min)
Your suggestions would be highly appreciated
The images that you see are the exact same samples run on two different occasions at exact same conditions
Thanks
Possible reasons:
The most likely reason is that your gels are expired or you have air spots trapped between the gel and plastic wall. That might happen when you press the gel between the plastic walls with your hands or when you remove the comb. It's better to make all manipulations with gels by holding the gels only from their edges.
Another possible reason is the presence of salts or detergents in your samples.
For western blots this observation might also point out to a air trapped between the gel and membrane during transfer step.
Possible reasons:
The most likely reason is that your gels are expired or you have air spots trapped between the gel and plastic wall. That might happen when you press the gel between the plastic walls with your hands or when you remove the comb. It's better to make all manipulations with gels by holding the gels only from their edges.
Another possible reason is the presence of salts or detergents in your samples.
For western blots this observation might also point out to a air trapped between the gel and membrane during transfer step.
Hi Aleks,
Apart from the suggestions given above by Dr. Viktor,, always flush the wells with the buffer before loading the samples...
bye
I know it is probably simple, but I've done it before...
Did you make sure to remove the green strips from the bottom of the gel? The precast gels have sticky strips on the bottom that you have to remove to get an even current.
If they are old, your stacking gradient may be bad too.
Hi Aleks,
You should see a paper clip icon labeled "File" just below the empty text box when you are editing either your question or your answer that will allow you to attach your gel photo.
In addition to suggestions above, the Bio-Rad trouble shooting guide lists other possibilities in this link:
http://www.bio-rad.com/en-us/applications-technologies/performing-protein-electrophoresis
If the link does not work, it's in the section of their website on Life Science Research, and is in the section entitled "Performing Electrophoresis". The problem is described as "skewed or distorted bands, lateral band spreading". From experience, the cause --high salt concentration-- does cause some bands to be wide, in which case, the band beside will be narrow. Another reason that they don't state is having a blank lane sometimes causes adjacent bands to be too wide. That can be prevented by preparing samples using just buffer and Laemmli sample buffer.
Assuming that you are controlling for everything else, the only remaining unknown is the gel itself. Why don't you try preparing it yourself instead of using precasts?
I agree with Alejandro.. Also make sure that your lower (separation) gel is completely polymerized before you move on to pouring the upper gel. Use freshly made APS and TEMED
I agree with Anna, the problem must be high salt concentration or that you leave a blank lane beside others. Try lowering the salt concentration of your samples and/or load the blank lanes just with buffer and Laemmli sample buffer.
Viktor and Carmen are correct. The banding pattern you described is from having too many ions or too few ions in the well. Rule number one is always load the same amount into each well. And make sure the ionic strength is roughly the same you can be off a little it will not make much of a difference. You have to watch your sugar content too. So when I say load the same amount I mean the same volume with the same amount of protein in the sample, example 10 ug protein in 25 uL of solution. Load every lane, make a dilution of you sample buffer and load it into empty wells.
I agree with myself, see above. If that does not work you my need to stack your proteins on the top of analytical gel. To do this, load your samples, see above, then run the gels at 10 mAmps constant amps not by volts. The dye marker and the proteins should stack up at the top of the analytical gel. When that has happened run you gel as usual.
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