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Biotinylation of protein in tris buffer


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#1 fancy_joule

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Posted 21 February 2011 - 11:56 AM

Hello,

I am currently attempting to biotinylate a protein, but the protein is dissolved in tris buffer (conc. is 0.476mg/ml). I attempted biotinylation of this protein a few weeks ago, and using the HABA/Avidin assay I calculated that there were 1.36 molecules Biotin/molecule CS. When I performed a BCA assay on my biotinylated protein, I did not get a positive result. We think that maybe the BCA assay wasn't sensitive enough, but we are also concerned that the tris buffer our protein is dissolved in competed with the biotin reaction.

I have been thinking about dialyzing the protein sample using Pierce's Slide-A-Lyzer cassettes (http://www.piercenet...4C-3E2075F80584) to remove the tris from the buffer. I have been using a biotinylation kit from Pierce (http://www.piercenet...?fldID=01030904).

Because I've repeated this assay multiple times with no success, I figure the issue must lie in the buffer. In the literature I've read, it seems that a similar kit has been used successfully. I am just wondering if anyone has had this problem, and what they did to get around it.

Any thoughts on this would be greatly appreciated! Thanks!

#2 braincow

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Posted 22 February 2011 - 10:21 AM

You can't use this kit with proteins in Tris buffer, and I believe it's stated in the manual. The Sulfo-NHS-biotin will react with the primary amines in Tris, interfering with your desired reaction. You'll have to exchange the buffer with PBS or something that doesn't have primary amines.

#3 fancy_joule

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Posted 03 March 2011 - 01:35 PM

You can't use this kit with proteins in Tris buffer, and I believe it's stated in the manual. The Sulfo-NHS-biotin will react with the primary amines in Tris, interfering with your desired reaction. You'll have to exchange the buffer with PBS or something that doesn't have primary amines.



Right. I knew this, which is why I thought about doing the buffer exchange using the Slide-A-Lyzer cassettes from Pierce.

Well I did the buffer exchange, and still am having no luck at all. I am using Pierce's HABA/Avidin Biotin Quantitation kit, and there is some biotinylation occurring. But when I do a BCA Assay, I am not getting a positive result.

Could it be that the BCA assay is not sensitive enough to detect my protein?

I think my next step will be doing affinity purification and seeing what happens. I have been working on this issue off and on for a month and feel like I am getting no where.




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