Hello,
I have been using in gel digestion of proteins and
conventional narrow bore HPLC for isolation of the proteolytically
derived peptides followed by Edman sequencing to identify unknown
proteins from SDS Gels. I have had mixed results over the last
several years. For example, sometimes a relatively faint coomassie
stained 80kDa band will yield good results but a smaller much more
heavily coomassie stained 30 kDa band will yield nothing.
I was interested in knowing what the experiences of others
using in-gel digestion has been. What is your overall success rate?
What enzyme gives you the most consistent results (good or bad)?
What is the relative sensitivity of the technique for example
relative to n-terminal sequencing of proteins transferred to PVDF?
And.... what do you tell investigators that comes to you with a
protein that needs gel purified as the final step of purification
prior to identification.... would you have the investigator stain the
gel for in-gel digestion, transfer to nitrocellulose for digestion on
the membrane or transfer to PVDF for n-terminal sequencing?
If there is interest, I can post a summary.
Thank you in advance for any and all input.
Brian Hampton
Project Leader, Hematopoiesis Department
American Red Cross, Holland Laboratory
15601 Crabbs Branch Way
Rockville, MD 20855
hampton@usa.redcross.org
Voice: 301-738-0814
Fax: 301-738-0811
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