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quantification of Biotinylated protein ??


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#1 rick112

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Posted 12 March 2009 - 11:41 PM

hi

I would like to know how can be quantify "Biotinylated protein" after Biotin labelling??

thanks a lot

#2 klinmed

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Posted 13 March 2009 - 03:08 AM

hi

I would like to know how can be quantify "Biotinylated protein" after Biotin labelling??

thanks a lot


You can use the HABA/avidin method. This is a very simple spectrophotometric technique and is available as a kit from Pierce (Pierce biotin quantitation kit). You can also use the method with solutions prepared in you own lab, much cheaper if you plan many determinations. The method, together with a protein determination (A280, BCA etc) will give you a value for the degree of biotinylation (mole biotin:mole protein). From this you can obtain an approximation of the conc of your biotinylated protein.

Hope this helps.

#3 rick112

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Posted 13 March 2009 - 11:05 PM

hi
thanks Klinmed...

will try it..just was curious how do you remove unlabeled biotin from the sample as free biotin could also give a reading...??
also does labelling with Biotin results in any charge, or hydrophobic difference to protein???

#4 klinmed

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Posted 13 March 2009 - 11:51 PM

hi
thanks Klinmed...

will try it..just was curious how do you remove unlabeled biotin from the sample as free biotin could also give a reading...??
also does labelling with Biotin results in any charge, or hydrophobic difference to protein???


Hi! Following labeling of your protein you should always remove free biotin. We usually do this by gel-filtration on disposable G25 columns (PD-10, GE Healthcare) or by VERY extensive dialysis (multiple buffer changes over 2-3 days). Biotin-labelling, in general, has minimal effects on the protein providing you donīt over-label. You should try to adjust the amount of biotinylation reagent so you only incorporate 3 -6 molecules of biotin per protein molecule.




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