As suggested, you can probably get good purification of heparin. If your pet protein has a known specific binding site, you can make it a personalized column by PCR'ing up arrays of directly repeated binding sites. The repeats will mis-anneal in subsequent rounds, giving rise to longer and longer products. If you use biotinylated primers, you can just stick the whole mess onto avidin beads. We've only been desperate enough to do this once, but it worked nicely. Phoebe
===================================== Phoebe A. Rice Dept. of Biochemistry & Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp ---- Original message ---- >Date: Sun, 10 Apr 2011 01:05:56 -0400 >From: CCP4 bulletin board <[email protected]> (on behalf of Zhijie Li ><[email protected]>) >Subject: [ccp4bb] immobilized DNA resin >To: [email protected] > >Hi, > >I have never done this myself, but as far as I know, DNA can be directly >conjugated through their primary amino groups to CNBr-activated beads or >NHS-activated agarose beads. These beads are supplied by many companies: >pierce, sigma, biorad, GE healthcare, etc.. - the same thing used for making >protein-conjugated beads through amine coupling. > >Unmodified DNA works, since the bases contain primary amines. In the early >days some people just loaded denatured DNA onto CNBr-activated beads and >then they would react. Here is one of the early papers: >http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1975.tb04151.x/pdf >My idea: if you generate sticky ends with some restriction enzymes or the >Klenow fragment, it should help exposing the bases on the overhangs, then >you should not need to denature the DNA and worry about all the crazy ways >that the DNA sits on the beads. > >Having said that, ideally, terminally amine-labeled oligos should be used >whenever possible, as it is more likely to gives you higher degree of >coupling and site-specific conjugation. Only one of the two chains needs the >NH2 label, then the two complementary oligos can be annealed to make a >one-end labeled dsDNA. When a longer piece is needed, the dsDNA can be >generated by PCR with one 5'amine labeled primer and one regular primer. You >can synthesize amine labeled oligos from most oligo synthesis facilities. > >Here is a literature discussing such coupling through amine modified oligos: >http://www.plosbiology.org/article/info:doi%2F10.1371%2Fjournal.pbio.0020173#pbio-0020173-g003 >Amine coupling is discussed in the part "Oligonucleotide Hybridization >Chromatography". > >Zhijie > >> >> -------------------------------------------------- >> From: "Alexandra Deaconescu" <[email protected]> >> Sent: Saturday, April 09, 2011 8:44 PM >> To: <[email protected]> >> Subject: [ccp4bb] immobilized DNA resin >> >>> Hello ccp4 enthusiasts: >>> >>> I am afraid this is a non-ccp4 related question. Can anyone recommend an >>> immobilized dsDNA chromatographic resin for purification of DNA-binding >>> proteins? GE seems to have something - I was wondering if people have >>> other recommendations? In the age of GST and His tags etc., these are not >>> very much used, but I do not have a tag in this case... >>> >>> Thanks a lot, >>> Alex >>
