dc984, on Sep 25 2009, 07:51 AM, said:
I've always used 1.5 mm, 10 well comb, but I've found that you need to load alot of sample (100 ug) for non-abundant proteins, otherwise the bands will be REALLY thin.
I was thinking, should I instead use a thinner 0.75 mm gel, 15 well comb, so that my Western bands will be "fatter"? What do you guys think? Is it always better to use smaller wells? Do you get lower sensitivity?
Thanks very much!
If you decrease the thickness of gel resistence will be more, in other words you can say same amount of charge will pass through the lesser area so better stacking of proteins you will get and you'll get sharper band. but the problem is with handling with thinner gel.
If you have around ten sample better to use 15 well comb and load your sample middle wells like 3rd to 12th to eliminate smiling effect. Second thing, number of combs have nothing to do with sensitivity.
Depth of the well is crucial for your results as you told that you are loading non-abundant protein it should not spill out. So depth of well should be enough to hold the sample.
hope it will help you.
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