Re: [ccp4bb] SDS and IMAC
Especially if you're dealing with lysate, I suspect the best way to do it is with magnetic Ni beads that you lift up and out of the gunk, to help avoid false positives from aggregating stuff that SDS/urea/guan would all elute. But why do you want X to remain on the column/beads? Removing Y but not X probably requires knowing more about the nature of their interaction than you do at this point? (e.g. will it take high salt, pH change, low (1M) urea, mild detergents etc) = 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: Thu, 23 Dec 2010 09:11:31 -0600 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Jacob Keller j-kell...@fsm.northwestern.edu) Subject: [ccp4bb] SDS and IMAC To: CCP4BB@JISCMAIL.AC.UK Dear Crystallographers, I am interested in doing a type of pull-down experiment by immobilizing protein X on IMAC resin, flowing a large volume of dilute lysate containing protein Y over it, then adding some concentrated agent (solid SDS perhaps) to some more of the same lysate, and running that over the column to elute protein Y off protein X, without eluting X off the column. I am afraid from past experience that SDS might knock X off the column, presumably depending on the concentration. I do not care about the folding state of Y--I will just be running a PAGE gel anyway. Does anyone know either what is the minimal concentration of SDS for robustly unfolding proteins/breaking up interactions (and whether that concentration is safe for IMAC), or what would be a good alternative agent to do the same? Thanks in advance for your help, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] SDS and IMAC
Use Urea - it does not interfere with gels etc. Additionally, you should consider covalent immobilization of protein X - using activated resins. Amine and carboxylic acid immobilization is common, however my all-time favorite is iodoacetamide-activated resin reacting with SH on the protein (if you have surface-exposed Cys - great, if you don't - just add one at a convenient spot, since your protein is recombinant anyway). While IMAC is a 'fairly strong' label, there will be cases where it leaches off, potentially complicating your analysis. Also you cannot reliably use it in the presence of quite a few chemicals that may be necessary for maintaining the competent state of protein Y. Also covalent modification allows you to elute with a variety of reagents including things that are not compatible with IMAC (e.g. low pH). May the Holiday Chipmunk regurgitate many presents under your Yuletide Shrub. Artem On Thu, Dec 23, 2010 at 9:11 AM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Dear Crystallographers, I am interested in doing a type of pull-down experiment by immobilizing protein X on IMAC resin, flowing a large volume of dilute lysate containing protein Y over it, then adding some concentrated agent (solid SDS perhaps) to some more of the same lysate, and running that over the column to elute protein Y off protein X, without eluting X off the column. I am afraid from past experience that SDS might knock X off the column, presumably depending on the concentration. I do not care about the folding state of Y--I will just be running a PAGE gel anyway. Does anyone know either what is the minimal concentration of SDS for robustly unfolding proteins/breaking up interactions (and whether that concentration is safe for IMAC), or what would be a good alternative agent to do the same? Thanks in advance for your help, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
[ccp4bb] SDS and IMAC
Dear Crystallographers, I am interested in doing a type of pull-down experiment by immobilizing protein X on IMAC resin, flowing a large volume of dilute lysate containing protein Y over it, then adding some concentrated agent (solid SDS perhaps) to some more of the same lysate, and running that over the column to elute protein Y off protein X, without eluting X off the column. I am afraid from past experience that SDS might knock X off the column, presumably depending on the concentration. I do not care about the folding state of Y--I will just be running a PAGE gel anyway. Does anyone know either what is the minimal concentration of SDS for robustly unfolding proteins/breaking up interactions (and whether that concentration is safe for IMAC), or what would be a good alternative agent to do the same? Thanks in advance for your help, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] SDS and IMAC
Hi Jacob Why not try with urea and for this type of studies I would probably use batch with the IMAC resin and not run the samples over a column. cheers Preben On 23/12/2010, at 16.11, Jacob Keller wrote: Dear Crystallographers, I am interested in doing a type of pull-down experiment by immobilizing protein X on IMAC resin, flowing a large volume of dilute lysate containing protein Y over it, then adding some concentrated agent (solid SDS perhaps) to some more of the same lysate, and running that over the column to elute protein Y off protein X, without eluting X off the column. I am afraid from past experience that SDS might knock X off the column, presumably depending on the concentration. I do not care about the folding state of Y--I will just be running a PAGE gel anyway. Does anyone know either what is the minimal concentration of SDS for robustly unfolding proteins/breaking up interactions (and whether that concentration is safe for IMAC), or what would be a good alternative agent to do the same? Thanks in advance for your help, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** J. Preben Morth, Ph.D Group Leader Membrane Transport Group Nordic EMBL Partnership Centre for Molecular Medicine Norway (NCMM) University of Oslo P.O.Box 1137 Blindern 0318 Oslo, Norway Email: j.p.mo...@ncmm.uio.no Tel: +47 2284 0794 http://www.ncmm.uio.no/research/ncmm-embl-group-leaders/
Re: [ccp4bb] SDS and IMAC
In my experience, either urea or guanidinium crashes out in gels. I can't remember--which one is it? I am thinking guanidinium. (If the answer to this email saves one grad student from the aggravation of such a phenomenon, it will have been worth it...) JPK On Thu, Dec 23, 2010 at 9:31 AM, Preben Morth j.p.mo...@ncmm.uio.no wrote: Hi Jacob Why not try with urea and for this type of studies I would probably use batch with the IMAC resin and not run the samples over a column. cheers Preben On 23/12/2010, at 16.11, Jacob Keller wrote: Dear Crystallographers, I am interested in doing a type of pull-down experiment by immobilizing protein X on IMAC resin, flowing a large volume of dilute lysate containing protein Y over it, then adding some concentrated agent (solid SDS perhaps) to some more of the same lysate, and running that over the column to elute protein Y off protein X, without eluting X off the column. I am afraid from past experience that SDS might knock X off the column, presumably depending on the concentration. I do not care about the folding state of Y--I will just be running a PAGE gel anyway. Does anyone know either what is the minimal concentration of SDS for robustly unfolding proteins/breaking up interactions (and whether that concentration is safe for IMAC), or what would be a good alternative agent to do the same? Thanks in advance for your help, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** J. Preben Morth, Ph.D Group Leader Membrane Transport Group Nordic EMBL Partnership Centre for Molecular Medicine Norway (NCMM) University of Oslo P.O.Box 1137 Blindern 0318 Oslo, Norway Email: j.p.mo...@ncmm.uio.no Tel: +47 2284 0794 http://www.ncmm.uio.no/research/ncmm-embl-group-leaders/ -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] SDS and IMAC
In my experience, either urea or guanidinium crashes out in gels. I can't remember--which one is it? I am thinking guanidinium. (If the answer to this email saves one grad student from the aggravation of such a phenomenon, it will have been worth it...) It's GuHCl and what crashes is dodecyl sulfate salts. Urea is fine (recall that many gels are run with urea in them and soem loading buffers contain urea). High salts is bad for gels but they are easy to remove from the samples by methanol/chloroform precipitation: http://www.abrf.org/ResearchGroups/EdmanSequencing/EPosters/ABRFhand1.doc (With low protein amounts, I'd suggest modifying this protocol by increasing centrifugation times: 10 min in the interphase forming step and 5-10 min in the last step of pelleting precipitated protein). Dima
