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 ***
Re: [ccp4bb] how to quantitate protein which dont have ne aromatic residue
Hi Arpita You can try QUANTI-iT Protein assay kit from Invitrogen. But still there is nearly 20-50% discrepancy between this method and a Abosorbance at 280. I also faced same problem with a protein, then re-cloned by adding a Trp at the C-terminus. Raj E. Rajakumara Postdoctoral Fellow Strcutural Biology Program Memorial Sloan-Kettering Cancer Center New York-10021 NY 001 212 639 7986 (Lab) 001 917 674 6266 (Mobile) --- On Sat, 9/4/11, Arpit Mishra ar...@igib.in wrote: From: Arpit Mishra ar...@igib.in Subject: [ccp4bb] how to quantitate protein which dont have ne aromatic residue To: CCP4BB@JISCMAIL.AC.UK Date: Saturday, 9 April, 2011, 3:22 PM hello everybody i am working on the protien which dont have any aromatic residue i do fplc other purification using 220 absorption, but i want to quantitate protein precisely i have tried using BCA nd bradford but both methods quantification is not matching,,so any one is having sum idea how to quantitate it precisely thanks in advance for your valuable suggestion.. Arpit Mishra
[ccp4bb] Used AREIMOL to judge the steric clashes
Hi, I have 2 rigid and fixed proteins and want to quickly judge whether there are some steric clashes. One quick way I am thinking is using CCP4 AREAIMOL to calculate the surfaces of each individual protein as well as the heterodimer, and check whether the sum of the two individual surfaces is larger then the dimer. I am wondering whether I can get some advices about this method. I also know there must be some other tools to quickly do it since this is kinda a simple docking problem, and I appreciate if suggested some more direct methods. Finally, I am also wondering whether AREAIMOL considers the assymetric unit during calculation. Thanks! Hailiang
Re: [ccp4bb] Used AREIMOL to judge the steric clashes
Areaimol is good for determining the contact area from the difference you mentioned. If you want to distinguish real clashes from comfortable van-der-Waals contacts, you can use pdbdist3: http://sb20.lbl.gov/berry/for/pdbdist3.for The two molecules have to be in separate pdb files. You give a threshold distance. For every atom in the first structure, every atom in the second structure that is closer than the threshold distance results in printing out the pair of atoms and the distance separating them. this gives a list of all contacts within the threshold distance. For v-d-w contacts are around 3.4 A, H-bonds 2.7, and anything closer than 2.0 could be considered a serious clash. Hailiang Zhang wrote: Hi, I have 2 rigid and fixed proteins and want to quickly judge whether there are some steric clashes. One quick way I am thinking is using CCP4 AREAIMOL to calculate the surfaces of each individual protein as well as the heterodimer, and check whether the sum of the two individual surfaces is larger then the dimer. I am wondering whether I can get some advices about this method. I also know there must be some other tools to quickly do it since this is kinda a simple docking problem, and I appreciate if suggested some more direct methods. Finally, I am also wondering whether AREAIMOL considers the assymetric unit during calculation. Thanks! Hailiang
Re: [ccp4bb] how to quantitate protein which dont have ne aromatic residue
Hi, You can read the spectrophotometric absorption at 280 nm and 200nm in UV range. It should serve your purpose and provide a decent idea for the amount of protein in the sample. Provided that absorbance at 280nm is given by aromatic rings but at the same time absorbance at 200nm is contributed by peptide bonds. A simultaneous reading at 260 nm which can be deducted later will substract the effect of nucleic acids. The relation between the readings at these values and protein concentration you can easily find anywhere on the web. Best wishes Gauri
[ccp4bb] O-linked glycosylation refinement in Phenix
Dear all,
I'm refining a structure which has both N-linked and O-linked
glycosylation. I use Phenix to do the refinement. It works well for the
N-linked NAG. I defined the link as the following:
apply_cif_link {
data_link = NAG-ASN
residue_selection_1 = chain A and resname NAG and resid 701
residue_selection_2 = chain A and resname ASN and resid 518
}
But it doesn't work when I defined the fucose-threonine link as the
similar way:
apply_cif_link {
data_link = FUC-THR
residue_selection_1 = chain A and resname FUC and resid 801
residue_selection_2 = chain A and resname THR and resid 596
}
The error message is missing CIF link: data_link_FUC-THR.
Does anyone have the experience of the O-linked glycosylaton refinement by
Phenix?
Thanks a lot!
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Re: [ccp4bb] Used AREIMOL to judge the steric clashes
Thanks Edward! Actually Areaimol works well for my problem. But now I have a new issue looking for some advice. I want to randomly generate some points in the unit cell and make a quick judgment whether it is outside of the solvent mask or not. It seems that Areaimol doesn't help at this point, and wonder whether some others tools in CCP4 can help to make it. Thanks again for your help! Best Regards, Hailiang Areaimol is good for determining the contact area from the difference you mentioned. If you want to distinguish real clashes from comfortable van-der-Waals contacts, you can use pdbdist3: http://sb20.lbl.gov/berry/for/pdbdist3.for The two molecules have to be in separate pdb files. You give a threshold distance. For every atom in the first structure, every atom in the second structure that is closer than the threshold distance results in printing out the pair of atoms and the distance separating them. this gives a list of all contacts within the threshold distance. For v-d-w contacts are around 3.4 A, H-bonds 2.7, and anything closer than 2.0 could be considered a serious clash. Hailiang Zhang wrote: Hi, I have 2 rigid and fixed proteins and want to quickly judge whether there are some steric clashes. One quick way I am thinking is using CCP4 AREAIMOL to calculate the surfaces of each individual protein as well as the heterodimer, and check whether the sum of the two individual surfaces is larger then the dimer. I am wondering whether I can get some advices about this method. I also know there must be some other tools to quickly do it since this is kinda a simple docking problem, and I appreciate if suggested some more direct methods. Finally, I am also wondering whether AREAIMOL considers the assymetric unit during calculation. Thanks! Hailiang
