| Quick navigation: | Home | Site Map || References | Biography || Copyright | Other copyright | Contact us | | |
|
|
Re: [ccp4bb] His tag does not bind. |
|
CCP4bb navigationCCP4bb <-- 2007 <-- October 2007 <-- 11 October 2007Subject: Re: His tag does not bind. From: Tim Gruene tg {- at -} SHELX {- dot -} UNI-AC {- dot -} GWDG {- dot -} DE Date: 2007-10-11 Qiagen's (or the other way round, depending on what you are using now). Qiagen use Ni-NTA while Pharmacia's resin is IDA which chelates Ni in a different way. During my PhD this made an all-or-nothing difference with a protein that expressed at several mg/l LB. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Wed, 10 Oct 2007, Das, Debanu wrote: > Hi, > This has been discussed often. You can look back through old posts or also look at troubleshooting pages in purification manuals that come with the some of the products like Qiagen Ni-NTA beads, etc. > > Briefly, you can try the following: > a) Play around with loading rate, i.e., go down to maybe 0.5 ml/min which may help to increase binding > b) If you are going by the back and adding 10 mM imidazole to the load, you can consider loading with no imidazole. > b) Add some urea to the load which may help to reveal the tag and make it bind better > c) Try some different salt concentrations in the loading buffer or no salt, also try some buffer or pH variation > d) Try adding some glycerol, 5-10%, which can help to reduce hydrophobic interactions, and may work if some impurity proteins are affecting target binding to column > e) You can try Talon resin instead of Ni > f) Try some ion-exchange column as the first step and then go back to metal chelating column. having a partially purified starting material may enhance binding to mc column. although your ion-exchange column will get dirty. > e) If these don't work, try C-terminal tag or different constructs with N-terminal tag > f) You could also try doing a denaturing prep and unfolding the protein. this should probably make it bind but refolding may be a problem for your protein size. however, if you have any assay set up and don't want to re-clone then you could give this a shot. > > > Regards, > Debanu. > > ________________________________ > > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of changrui lu > Sent: Wednesday, October 10, 2007 6:13 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] His tag does not bind. > > > Dear all, > > I am trying to express a 150 kd protein in E coli. I have it in two constructs, one with pmal-his and other with only his tag at N terminus. The full length protein can be detected both by sds and western using anti-his (190kd and 150kd respectively) but strangely neither binds to his-column very well. The majority of the full length comes through the column either at loading step or low salt wash step. The major species that gets trapped and eluted is the mbp-his truncation (~40kd). Some, though very little, full length protein did make it out the his column. The pmal-his construct does not bind amylose resin any better with majority flows right through. All purification are carried out under standard conditions as mentioned in the manuals. The protein is soluble and does not precipitate in the columns. I appreciate and ideas or explanations. > > Thanks in advance. > > Ray > Cornell Univerisity > > CCP4bb navigationCCP4bb <-- 2007 <-- October 2007 <-- 11 October 2007 |
| ProteinCrystallography.org: Copyright 2006-2007 by Quid United Ltd |