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Re: [ccp4bb] Offtopic - In vitro protein interaction analysis using Diazirine crosslinkers.
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Subject: Re: Offtopic - In vitro protein interaction analysis using Diazirine crosslinkers.
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2009-11-04

Dear Rohan,



Your most significant concern (in my opinion) should be the nature of the
protein - at 9kDa with only two primary amines (of which one is the
N-terminus, I assume?) you're dramatically changing the properties of the
protein when you label it. Depending on which of the crosslinkers you're
using, you are removing both charges and at the same time adding
considerable amount of 'grease' to the surface. It's therefore not too
surprising that the protein crashes out.



Can you use more hydrophilic crosslinkers? What are you trying to
accomplish?



Cheers,



Artem



_____

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Rohan
Agashe
Sent: Wednesday, November 04, 2009 8:55 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Offtopic - In vitro protein interaction analysis using
Diazirine crosslinkers.



Apologies for an off topic question. I was hoping for some advice regarding
the use of sulfo-NHS diazirine crosslinker (Pierce link
473A6F2468A1> ) for in vitro protein interaction analysis.

Have tried labeling the protein (9kDa, 10 to 20uM conc) with 2 primary
amines using 30-40X excess Sulfo-NHS-Diazirine crosslinker in PBS (1hour at
4deg). Have tried other concentration combination as well, but this ratio
yields some usable material. However, majority of the protein crashes out of
solution.

Was wondering if anyone has any suggestions in optimizing the yield of
crosslinker labelled protein?

Thanks in advance.

--
Rohan Agashe
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