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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:[log in to unmask]] On Behalf Of Rohan Agashe Sent: Wednesday, November 04, 2009 8:55 AM To: [log in to unmask] 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 <http://www.piercenet.com/products/Browse.cfm?fldID=DB013E61-5056-8A76-4EC4- 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