Protein drag in IEF-WB - (Dec/16/2009 )
Hi all... what do you all think this drag is due to??
i thought it may be due to salt.. but then it is giving me color with the substrate... and my antibodies are very specific so it cant be anything else too.... i need ideas... i m anyways going to desalt and run the samples in water to address the salt thing... any other ideas??!!!
looks like slowly dissolving aggregates of your protein.
mdfenko on Dec 16 2009, 09:05 PM said:
oh!!! but my protein does not have any aggregated impurity and moreover i run a denaturing urea gel.... i load around 3 mcg. I know tat can be too much for a WB application but the titre of my antibody is low so i have to load this much!!!
even if its aggregates what corrective action do u suggest??
are you boiling your samples prior to loading? if so, how long?
too long can actually cause aggregation.
you could try heating your samples at 65-70C for 10 minutes (or more or less, as you determine necessary).
also, make sure you have enough reducing agent around, to prevent re-bonding (or you can treat with iodoacetamide).
also, with aggregates, sometimes you can't fully solubilize them. you may need to clarify the sample (by centrifugation or filtration or both).