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Hi, Typically one does not expect massive contamination with DNA unless the protein you work with is either very basic, or is a DNA/nucleotide binding protein. Having said this, I must add that in almost every IMAC preparation that I have *ever* run, regardless of the resin used, there was always a small but detectable amount of nucleic acid together with the protein. This becomes very evident in the second step which often is done by ion exchange using some form of a quaternary-amine resin. The nucleic acid separates very nicely and elutes as a separate peak at around 35 mS/cm (where very few proteins still stick to the resin) with 260/280 ratio > 1.50. What you have, assuming that the third peak is predominantly protein, is kind of like that - the first peak is probably DNA aggregates (cross-stitched by various 'sticky' proteins). Not sure about the second peak - I don't know what column was used and where its void volume might be, etc. So, in conclusion - DNA contamination of IMAC preps is real and common, the amounts vary - if you have abundant protein of interest and you're using a correct ratio of protein to resin, the amount of DNA would be less, however if you're using a large excess of resin to protein (and/or your protein is just isn't abundant), the DNA amounts can be comparable to that of protein. As you mention, it does not take much DNA to overpower the protein signal, since the specific absorption of DNA is usually much higher than that of (most) proteins. Two easy methods seem to minimize DNA contamination of 'regular' protein preps - a) washing the column with 1M NaCl prior to elution or b) using ion-exchange as the second step. In special cases it may be very hard to resolve the DNA away, and in some hellish cases (like one of the proteins I am working on right now) the removal of DNA resuts in irreversible precipitation of the protein of interest. To detect DNA you could try staining with SYBR green or something like that. Good luck, Artem _____ From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Jacob Keller Sent: Tuesday, January 08, 2008 4:20 PM To: [log in to unmask] Subject: [ccp4bb] DNA contamination post-Talon? Sorry for the off-topic question again, but has anybody encountered DNA fragments' sticking to a Talon column? I got the attached trace after putting my Talon-purified protein onto an s75 column. Notice that the A254:A280 ratio changes pretty dramatically from the first peak to the last (although the scales are off a bit--the red trace should be about half what it is. This actually happened quite a while ago, so there are some experimental gaps (such as a coomassie gel of all fractions), but from the post-Talon gel, the protein looks about, say, 90% pure (probably better)? I am trying to make sense of this in light of some more recent developments. Could it be a contaminating protein bound to nucleotide or some other highly-absorptive small molecule in the void volume? I would not think that would have enough absorbance, really, to be seen so prominently. Or perhaps fragments of DNA, which have a much greater E254, and might run in the void volume if sufficiently large, but I would not think they would make it past the Talon (hence the question)...Any suggestions welcome...Also, from the dried coomassie gel, could I determine whether there is DNA there? How can one detect DNA in PAGE gels? Jacob Keller ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [log in to unmask] ******************************************* ----- Original Message ----- From: Avinash Gill <mailto:[log in to unmask]> To: [log in to unmask] Sent: Tuesday, January 08, 2008 2:42 PM Subject: Re: [ccp4bb] Any programs other than GRASP have a surface scribing function? I have used CCP4MG to produce a molecular surface of the model that i have been working on. There is an example in the online documentation to produce a molecular surface for the entire molecule or for a subset of atoms, and color it by solvent accessibility or other properties. It produces beautiful images, although if you do not have a relatively fast processer, computation of the surface might slow your machine temporarily. The have been able to produce these molecular surfaces in CCP4MG running on a Windows Vista machine. Hope this helps. Avi. On Jan 8, 2008 3:01 PM, James Thompson < [log in to unmask] <mailto:[log in to unmask]> > wrote: Dear CCP4'ers, Any Windows/Linux/OS X programs have a surface scribing function similar to that found in GRASP, to quickly draw the border of a surface selection (or subset) and calculate or display local features of the molecular surface? I have no access to SGIs now. I have a memory of similar function elsewhere but am not finding the ability within GRASP2, DINO, CHIMERA, etc. Perhaps I'm thinking of Setor which was also IRIX, and perhaps this memory is suspect... Other selection methods for a molecular surface require more of my time to define the subset. Many thanks, Jim James R. Thompson Assistant Professor of Biophysics Mayo Clinic College of Medicine Department of Physiology and Biomedical Engineering Mayo Proteomics Research Center Office 507-538-3891 Fax 507-538-3954 E-mail [log in to unmask]