gradient sds-page.. how would that improve resolution? - (May/14/2008 )
hi all.. i agree that gradient sds-page r better used when u want to have a wide range mol wt proteins in a gel.. i.e. high to low mol wt proteins in the same gel. but if i have a dgradation product which is near my protein molecular weight.. would running a gradient help better resolution? i dont think so. pls tel if u think its a yes..
what r the kda values that can be differentiated max in an sds-page.. vs gradient sds-page. which is better resolving.
while you are correct that gradient sds page is best suited for showing high and low mw proteins in the same gel it still helps resolution in a couple of ways.
first, by getting more and more restrictive as the protein moves down the gel, it helps maintain stacking. sharper bands won't overlap as much (unless they are the same).
second, by engineering the gradient properly you can enhance the separation of closely moving bands. we have run shallow gradients (5% or less difference) to enhance the separation of close moving bands (sometimes we also add urea or a gradient of urea opposite to the acrylamide gradient).
but, we have also seen baseline resolution with three bands that are 17, 17.5 and 18 kDa on 5-15% gradients (disclaimer: the gels are large and we use the neville buffer system rather than laemmli).
I agree with menfenko. Sharper bands and broad ranges are two advantages for gradient gels over homogeneous gels.