sample preparation from whole cell lysates for Edman degradation
Posted 18 September 2012 - 02:19 AM
I want to send one protein for Edman degradation, but i don't have it purified. Since it should be 1microgram/lane I was wondering is there any way to determine amount of protein of interest from SDS/PAGE gel or PVDF membrane? I was thinking of doing Western blot on one half on the membrane and when I detect band of interest, I would cut the same band on the other half of the membrane which I didn't use for Western blot, but I still don't have any idea for determining the amount of my protein in that particular band. I can't use chromatography, mass spec etc I need something that would save me money, if that's possible.
So, anybody had experience with this? Thank you in advance!
Posted 18 September 2012 - 04:39 AM
An easy approach might be to :
- run your sample in duplicate on a gel
- divide the gel in half
- coomassie stain one half, do your transfer with the other half
- do densitometry on your coomassie stained half to know how much of your POI is there
- proceed with edman degradation with the transferred half based on your findings from the coomassie stained half
Posted 18 September 2012 - 04:57 AM