Re: [ccp4bb] immobilized DNA resin
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 CCP4BB@JISCMAIL.AC.UK (on behalf of Zhijie Li zhijie...@utoronto.ca) Subject: [ccp4bb] immobilized DNA resin To: CCP4BB@JISCMAIL.AC.UK 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./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 deac...@brandeis.edu Sent: Saturday, April 09, 2011 8:44 PM To: CCP4BB@JISCMAIL.AC.UK 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
Re: [ccp4bb] immobilized DNA resin
Heparin simulates the structure of DNA and RNA, so it has nonspecific affinity towards DNA or RNA binding protein. It has also been used as DNAse or RNase inhibitor but it is not very good one. Nian Huang, Ph.D. UT Southwestern Medical Center On Sat, Apr 9, 2011 at 7:44 PM, Alexandra Deaconescu deac...@brandeis.eduwrote: 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
Re: [ccp4bb] immobilized DNA resin
I wonder whether that is why it works as an anti-coagulant? Is it mimicking nucleic acids? So then would DNA work as an anti-coagulant as well...? JPK On Mon, Apr 11, 2011 at 4:20 PM, Nian Huang huangn...@gmail.com wrote: Heparin simulates the structure of DNA and RNA, so it has nonspecific affinity towards DNA or RNA binding protein. It has also been used as DNAse or RNase inhibitor but it is not very good one. Nian Huang, Ph.D. UT Southwestern Medical Center On Sat, Apr 9, 2011 at 7:44 PM, Alexandra Deaconescu deac...@brandeis.edu wrote: 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 -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] immobilized DNA resin
Hi Alex, as you have a DNA binding protein does it have a high pI? If so, good old ion exchange with an S-sepharose column should do it. Cheers James Dr James Garnett Division of Molecular Biosciences, Imperial College London, Level 5, Biochemistry Building, South Kensington, LONDON SW7 2AZ, UK. Tel: +44 (0) 207 594 5464 Fax: +44 (0) 207 594 3057 - Reply message - From: Raji Edayathumangalam r...@brandeis.edu To: CCP4BB@JISCMAIL.AC.UK CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] immobilized DNA resin Date: Sun, Apr 10, 2011 03:38 Hi Alex, Most DNA-binding proteins has decent affinity to heparin columns. Have you tried purifying your protein over heparin columns? Cheers, Raji On Sat, Apr 9, 2011 at 8:44 PM, Alexandra Deaconescu deac...@brandeis.edumailto:deac...@brandeis.edu wrote: 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 -- --- Raji Edayathumangalam Research Fellow in Neurology, Harvard Medical School Postdoctoral Fellow, Center for Neurologic Diseases, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] immobilized DNA resin
Related fact I learned recently, perhaps of interest for fellow record-keepers: heparin sulfate has the highest negative charge density of any known biological molecule. JPK On Sat, Apr 9, 2011 at 9:38 PM, Raji Edayathumangalam r...@brandeis.edu wrote: Hi Alex, Most DNA-binding proteins has decent affinity to heparin columns. Have you tried purifying your protein over heparin columns? Cheers, Raji On Sat, Apr 9, 2011 at 8:44 PM, Alexandra Deaconescu deac...@brandeis.edu wrote: 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 -- --- Raji Edayathumangalam Research Fellow in Neurology, Harvard Medical School Postdoctoral Fellow, Center for Neurologic Diseases, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] immobilized DNA resin
heparin sulfate has the highest negative charge density of any known biological molecule. Seems to me that phytic acid (IP6, C6H6-(H2P04)6) and inositol heptakisphosphate (IP7) should have higher charge per mass and per volume. - Dima
Re: [ccp4bb] immobilized DNA resin
Hey, does Wikipedia lie? Also, the reference is to Lehninger's text, although I did not verify it. http://en.wikipedia.org/wiki/Heparin On Sun, Apr 10, 2011 at 3:37 PM, Dima Klenchin klenc...@facstaff.wisc.edu wrote: heparin sulfate has the highest negative charge density of any known biological molecule. Seems to me that phytic acid (IP6, C6H6-(H2P04)6) and inositol heptakisphosphate (IP7) should have higher charge per mass and per volume. - Dima -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
[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
Re: [ccp4bb] immobilized DNA resin
Hi Alex, Most DNA-binding proteins has decent affinity to heparin columns. Have you tried purifying your protein over heparin columns? Cheers, Raji On Sat, Apr 9, 2011 at 8:44 PM, Alexandra Deaconescu deac...@brandeis.eduwrote: 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 -- --- Raji Edayathumangalam Research Fellow in Neurology, Harvard Medical School Postdoctoral Fellow, Center for Neurologic Diseases, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] immobilized DNA resin
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./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 deac...@brandeis.edu Sent: Saturday, April 09, 2011 8:44 PM To: CCP4BB@JISCMAIL.AC.UK 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
