Quality of aliquot for SDS-page in protein purification - When is pure pure enough? (Aug/28/2009 )
I wanted to know what the standard was in considering something a protein sample as pure. Everyone tells you to take an aliquote of ur fractions and load them on a gel to check for purity. The thing is I used to elute my protein in 1ml fractions and loaded 10ul on a gel. Saw no contaminants after tweeking the protocol, was very happy. Then I decided to elute in 500ul fractions and load as much as I could on the gel. So I ended up without actually wanting to loading 40-50ug of my protein on gel. Surprise surprise I see several bands in the background. So what should I gain from this observation? Am I just being too anal about this? Could someone maybe tell me what the standard is for purity because I really dont get it anymore but I want to be sure that my work is good.
Im sorry if this is a stupid post but I dont like the idea of not knowing and I hope you are all looking forward to the weekend like I am
Thank you in advance!
98-99% purity is pretty good. eventually you will reach the point of diminishing returns, where the cost in yield is too high to justify a small gain in purity.
the protein needs to be pure enough that the contaminants won't significantly affect your results.