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Western Blot: Gel Thickness

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Western Blot: Gel Thickness

Postby earmst03 » Tue Aug 21, 2007 9:49 pm

What effect does gel thickness (ie 0.5mm vs 1.0mm) have on the running and transfer times and voltages? Does the thinkness influence the quality of the WB, detection, or imaging? Anything else important about gel thickness? Thanx!!!
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Postby blcr11 » Wed Aug 22, 2007 8:15 pm

I’m not absolutely certain about this. The electrophoretic mobility of ions in an electical field are more strongly influenced by the surface area than by the path length, per se. I’m not sure which way you’re trying to go (from thicker to thinner or the other way around) but so long as the buffer composition is the same, your run conditions shouldn’t have to change too much. If I were going to thicker gels where I’d normally run thinner ones, I suppose I would increase the run time a little to give the proteins more time to exit the gel and bind to the membrane, but I wouldn’t overdo it, either. If I were going to thinner from thicker, I think I would just leave the run conditions alone unless I had reason to believe I was in danger of running proteins completely through the membrane (a rare problem, and more likely for small peptides than proteins much greater than, say 3-5 kD). I don’t think I would try to change the voltage or current unless the current drops to near zero (but then I would look carefully to see that the apparatus was OK and the buffers were made correctly, etc, before I goosed the voltage much).

The main advantage of 1 mm over 0.5 mm gels is the increase in load volume for the thicker gel; the sensitivity of the western in increased by the fact that you can load twice as much “stuff” per lane without having to concentrate the sample. There in a minor trade-off in resolution as the bands from 1 mm gels may be a little wider than the same band from a 0.5 mm gel, but this is usually not enough of a problem to worry about, and if it makes the difference between seeing or not seeing a band, it is worth it.
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