Biotinylation of Ab

BiotecBurd biotecburd at aol.com
Sun Sep 11 11:38:04 EST 1994


In article <34r039$5rn at search01.news.aol.com>, gdwx at aol.com (GDWX) writes:

Dig is short for digoxigenin, a plant steroid found in foxglove. 
Boehringer Mannheim produces a series of kits using this antigen as a
marker (label) which is detected using a anti-dig polyclonal produced in
sheep.  However they did it, the Fab fragment is very "non-sticky".  When
using biotinylated Ab or probes, there is always the difficulty of
indigenous biotin which can create background when streptavidin-enzyme
conjugates are used to detect the bioinylated marker.  As long as you are
not working with foxglove (and who is) indigenous digoxigenin does not
exist.  

The dig-NHS molecule I mentioned is simply activated digoxigenin, much as
biotin-NHS is used to label proteins, or more specifically, primary
amines.  Alternative dig-derivatives can be used to label free SH groups. 


The primary use of dig is with DNA probes, although I have successfully
used it with RNA, especially in Northern blots and in situ hybridizations.
 It can also be used to label antibodies, and thus can be used in a
variety of immunoassays.  Anti-dig Ab can be purchased which has alkaline
phosphatase or HRP as an enzyme label, or with a variety of fluorescent
markers.

This system is definitely worth trying.  Good luck.

Dave Burden
Biotechnology Training Institute
Bridgewater, NJ



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