Re: [ccp4bb] Quality of His-Select Resin After Regeneration
I use a 10 ml GE-healthcare Ni-sepharose FF column, packed myself. I strip it with EDTA after a run and then recharge before the next one. It's has probably been used 30+ times, and I have no plans or repacking it anytime soon, it still works as good as it ever did. We purify secreted proteins from insect cells, so it's a pretty clean sample. If you use lysates I would imagine you would need more intensive cleaning with detergents and denaturants. But if the resin still turns blue (or pink for Co) it ought to work fine in my opinion, unless the frits are clogged, or a large amount of precipitation is trapped in the beads. I would unpack the column and wash the resin in batch mode in that case. Nat On Fri, Jul 23, 2010 at 1:20 AM, Artem Evdokimov ar...@xtals.org wrote: Hi, His-Select has been my favorite resin for IMAC for as long as Sigma's been selling it. It's not the cheapest resin but at roughly $11/ml packed bed volume and a capacity of ~10-20 mg protein per ml of resin bed (depends on protein size, quality, and other factors) there seems to be little point in regenerating the resin beyond 1-2 times. In other terms, even at single-use it's still a good value since cutting down on purification stages and duration (without sacrificing protein quality!) is a certain way to increase useful protein yield and (in my opinion) the likelihood of crystallization... I've also noticed that recycled resin gives somewhat more contaminants (which is true for pretty much any brand of IMAC media) and since 10-15mg of first-stage protein goes a long way in our lab I do not feel particularly bad when I sacrifice a couple of ml of resin to be sure that my protein is as good after primary separation as it can ever be. Cobalt loading in my hands did not result in improved quality as the resin binds very few contaminants by itself if used appropriately. Primary contaminants arise from (a) sub-optimal coverage of binding sites by the target protein and (b) from unwanted 'passengers' riding on your protein of interest. The former can be minimized by dialing the protein/resin ratio such that the protein is in slight excess (i.e. resin can be saturated) and the latter can often be avoided by proper selection of lysis and purification buffers (pH, ionic strength, cosolutes, detergents and so forth). Your mileage will vary, especially with membrane proteins or hideous sticky/aggregated monsters like those that seem to frequent my lab way more often than I like... The HF form of this resin is very convenient for semi-batch separations (i.e. lysis and incubation with resin in batch, followed by washing and elution on a column of some sort). This way a number of proteins can be quickly purified in parallel without any special equipment - from a half dozen 1-5ml columns to a 96-well filter plate on a suction or centrifugal separator. Artem P.S. since you ask, depending on the brand of GSH resin it also can survive a few regeneration cycles -- but again depending on various factors (mostly on what kind of lysates you're loading) the quality and quantity of eluted protein(s) will inevitably decline with use. On Thu, Jul 22, 2010 at 9:05 PM, Matthew Bratkowski mab...@cornell.edu wrote: Hi. We have been using His-Select Resin from Sigma in our lab for a number of years now. When we first bought the resin, I usually got much better purity of His tagged proteins compared with regular Ni-NTA resin. However, after regenerating the resin several times, the level of purity seems to have declined. Has anyone else noticed this with His Select? In general, could someone suggest the typical lifespan of His Select or Ni2+ resin in general? What about Glutathione resin? I was also wondering if anyone had experience using cobalt resin? What is the binding capacity of cobalt compared to nickel, and is the selectivity any better than either His-Select or regular Ni-NTA? Also, is it possible to just strip nickel off of the resin and then recharge it with cobalt? Thanks, Matt
[ccp4bb] Quality of His-Select Resin After Regeneration
Hi. We have been using His-Select Resin from Sigma in our lab for a number of years now. When we first bought the resin, I usually got much better purity of His tagged proteins compared with regular Ni-NTA resin. However, after regenerating the resin several times, the level of purity seems to have declined. Has anyone else noticed this with His Select? In general, could someone suggest the typical lifespan of His Select or Ni2+ resin in general? What about Glutathione resin? I was also wondering if anyone had experience using cobalt resin? What is the binding capacity of cobalt compared to nickel, and is the selectivity any better than either His-Select or regular Ni-NTA? Also, is it possible to just strip nickel off of the resin and then recharge it with cobalt? Thanks, Matt
Re: [ccp4bb] Quality of His-Select Resin After Regeneration
The Agarose superflow material is I think the same for Talon (Clontech) and Qiagen, not sure about Machery Nagel or Sigma's product. Superflow agarose is cross-linked differently than normal sepharose/cross-linked agarose. The nature of chelates and their attachments also differ. Qiagen, Sigma and Pharmacia use NTA while Clontech (Talon) uses carboxymethyl aspartate. Qiagen uses original chemistry through lysine spacer, Sigma's synthetic route is through bis-carboxymethyl cysteine (or -cystine), and Pharmacia opted for a clean but complex chemistry that results in a very hydrophilic spacer and higher degree of derivatization. Dima
Re: [ccp4bb] Quality of His-Select Resin After Regeneration
Hello Matthew, I almost always charge my IMAC resin with Co instead of Ni. I find that it gives much better purity because the selectivity is better. This is because the coordination sphere around the Co atoms require more His residues than the Ni coordination sphere. I believe Co requires 4 His, while Ni only requires 2. That being said, although it is more selective, proteins with a His tag are likely to have lower affinity for Co, and will therefore elute in lower imidazole concentrations, and if your His tag is not entirely exposed, you may have issues getting it to bind at all. I have never had this problem, but I have heard that it can be an issue. It is probably worthwhile to try cobalt, but do it under the assumption that there is a small chance you will have to go back to Ni. Also, if you use cobalt, you may notice a slight color change in the column when you run it under certain buffer conditions. Some additives cause the color to change from light pink to a purplish color. This is ok, the column will still bind your protein fine. Just be sure to look in the resin manufacturer's manual to make sure you don't have any incompatible components in your buffer. In general, anything that can go over the column when it is charged with Ni should be ok for Co as well. Good luck, Mike Thompson - Original Message - From: Matthew Bratkowski mab...@cornell.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, July 22, 2010 7:05:21 PM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] Quality of His-Select Resin After Regeneration Hi. We have been using His-Select Resin from Sigma in our lab for a number of years now. When we first bought the resin, I usually got much better purity of His tagged proteins compared with regular Ni-NTA resin. However, after regenerating the resin several times, the level of purity seems to have declined. Has anyone else noticed this with His Select? In general, could someone suggest the typical lifespan of His Select or Ni2+ resin in general? What about Glutathione resin? I was also wondering if anyone had experience using cobalt resin? What is the binding capacity of cobalt compared to nickel, and is the selectivity any better than either His-Select or regular Ni-NTA? Also, is it possible to just strip nickel off of the resin and then recharge it with cobalt? Thanks, Matt -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] Quality of His-Select Resin After Regeneration
Hi, His-Select has been my favorite resin for IMAC for as long as Sigma's been selling it. It's not the cheapest resin but at roughly $11/ml packed bed volume and a capacity of ~10-20 mg protein per ml of resin bed (depends on protein size, quality, and other factors) there seems to be little point in regenerating the resin beyond 1-2 times. In other terms, even at single-use it's still a good value since cutting down on purification stages and duration (without sacrificing protein quality!) is a certain way to increase useful protein yield and (in my opinion) the likelihood of crystallization... I've also noticed that recycled resin gives somewhat more contaminants (which is true for pretty much any brand of IMAC media) and since 10-15mg of first-stage protein goes a long way in our lab I do not feel particularly bad when I sacrifice a couple of ml of resin to be sure that my protein is as good after primary separation as it can ever be. Cobalt loading in my hands did not result in improved quality as the resin binds very few contaminants by itself if used appropriately. Primary contaminants arise from (a) sub-optimal coverage of binding sites by the target protein and (b) from unwanted 'passengers' riding on your protein of interest. The former can be minimized by dialing the protein/resin ratio such that the protein is in slight excess (i.e. resin can be saturated) and the latter can often be avoided by proper selection of lysis and purification buffers (pH, ionic strength, cosolutes, detergents and so forth). Your mileage will vary, especially with membrane proteins or hideous sticky/aggregated monsters like those that seem to frequent my lab way more often than I like... The HF form of this resin is very convenient for semi-batch separations (i.e. lysis and incubation with resin in batch, followed by washing and elution on a column of some sort). This way a number of proteins can be quickly purified in parallel without any special equipment - from a half dozen 1-5ml columns to a 96-well filter plate on a suction or centrifugal separator. Artem P.S. since you ask, depending on the brand of GSH resin it also can survive a few regeneration cycles -- but again depending on various factors (mostly on what kind of lysates you're loading) the quality and quantity of eluted protein(s) will inevitably decline with use. On Thu, Jul 22, 2010 at 9:05 PM, Matthew Bratkowski mab...@cornell.edu wrote: Hi. We have been using His-Select Resin from Sigma in our lab for a number of years now. When we first bought the resin, I usually got much better purity of His tagged proteins compared with regular Ni-NTA resin. However, after regenerating the resin several times, the level of purity seems to have declined. Has anyone else noticed this with His Select? In general, could someone suggest the typical lifespan of His Select or Ni2+ resin in general? What about Glutathione resin? I was also wondering if anyone had experience using cobalt resin? What is the binding capacity of cobalt compared to nickel, and is the selectivity any better than either His-Select or regular Ni-NTA? Also, is it possible to just strip nickel off of the resin and then recharge it with cobalt? Thanks, Matt
