Re: [ccp4bb] Protein elution in Size Exclusion

[Roger Rowlett]

I'm glad this worked out well for you. For conducting molecular sizing determinations with a calibrated GEC column, I typically use 100-500 uL injections of approximately 1 mg/mL or so of purified protein on a 1.6 x 60 cm column. The limited protein quantity will prevent saturation of the internal space of the GEC media, and the limited concentration minimizes vicosity effects which hinder mass transfer between the stationary and mobile phases. At least 100 mM NaCl is important to prevent residual ion exchange interactions with the gel medium. I once worked with a protein that completely "disappeared" on a Sephadex column--lots of protein in, no protein out--at low ionic strength. When the column was washed with 500 mM NaCl, the protein magically re-appeared. Usually 100 mM NaCl is sufficient to prevent this occurrence. Interestingly, I had a very similar conversation about GEC with a colleague and research collaborator just a couple of weeks ago. The apparent MW of our protein sample was all over the map until the sample size, concentration, vicosity, and eluant ionic strength were properly controlled, then the apparent measured MW was consistently within 10% of the calculated mass of the holoenzyme suggested by crystallography. This is typical. Keep in mind GEC measure molecular VOLUME and not MW, but most of the time we make certain assumptions that make apparent MW measurements "close enough for government work." Pay attention to the exclusion limits of the medium as well. Proteins that elute close to the void and total volumes will have poor precision in either the calibration curve or unknown determination. Cheers, _______________________________________ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 8/30/2011 2:50 PM, Allan Pang wrote: Thanks everyone for your response. The most likely answer to my problem is protein overloaded onto the column. I pushed my protein concentration further down to 0.5ml instead of the usual method, which to run multiple times on SEC. Adding NaCl in the buffer may also help, as it seems that the broadness of the elution was affected by addition of NaCl. I don't think the column is knackered because the previous (and succeeding) runs of other protein samples were okay. Different -mer state is a possibility, but something that I am not really leaning towards to, since, I managed to purify the protein before in a good elution size. Science mystery solved. Thanks again! Allan Quoting Filip Van Petegem <filip.vanpete...@gmail.com>: worst-case scenario for crystallization purposes: 9. Your protein runs as a mixture of monomers, dimers, trimers, whatever-mers... Filip On Sun, Aug 28, 2011 at 7:24 AM, David Briggs  <drdavidcbri...@gmail.com>wrote: Following on from Roger's fine suggestions: 8. Your column is knackered. Can you see fine lines or cracks in the column? Good packing is v.important for SEC columns. HTH, Dave ============================ David C. Briggs PhD Father, Structural Biologist and Sceptic ============================ University of Manchester E-mail: david.c.bri...@manchester.ac.uk ============================ http://manchester.academia.edu/DavidBriggs (v.sensible) http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB ============================ On 28 August 2011 10:25, Allan Pang <a.p...@qmul.ac.uk> wrote: > Hi there everyone, > > What does it mean when you have proteins eluting in almost the whole column > volume of S200? > > I ran a gel with fractions from 8ml to 20ml and saw band for my protein all > throughout. > > Judging peaks on chromatogram is not useful as it doesn't have any aromatic > rings. > > Cheers, > > Allan > > -- > Allan Pang > > PhD Student > > G35 Joseph Priestley Building > Queen Mary University of London > London > E1 4NS > > Phone number: 02078828480 > -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/