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sample preparation from whole cell lysates for Edman degradation


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#1 suncokret

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Posted 18 September 2012 - 02:19 AM

Hello!

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!

#2 proteaMatt

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Posted 18 September 2012 - 04:39 AM

Do you have access to a Gel Doc or some other gel imaging system? I know with the system we have you can perform densitometry on coomassie stained gels. If you don't have access to a machine like that, you might be able to find another method to perfom densitometry using equipment you do have access to. Perhaps a google (scholar) search for densitometry and SDS-PAGE could get you started.

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
I don't have the details on all of this, because I don't have much experience with it, but hopefully this can at least point you in the right direction to start looking.
Lab Technician at Protea Biosciences

#3 suncokret

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Posted 18 September 2012 - 04:57 AM

Thank you so much! I have access to GelDoc and I will follow your advice :)




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