Re: [ccp4bb] His tag does not bind.

2007-10-11 Thread Tommi Kajander
bulletin board [mailto:[EMAIL PROTECTED] 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

Re: [ccp4bb] His tag does not bind.

2007-10-11 Thread Anastassis Perrakis
Let me make three short notes: 1. If the protein does not bind in a 'normal' buffer try first under denaturing conditions to see if everything is expressed correctly (a repeat of Artem's suggestion basically!). Protocols for that are in all materials manuals. 150 kD in E.coli is very very tou

Re: [ccp4bb] His tag does not bind.

2007-10-11 Thread Chun Luo
bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jacob Keller Sent: Thursday, October 11, 2007 10:03 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] His tag does not bind. Recently, a make-or-break difference for me was making sure the pH was what I thought it was: In the presence of 50mM HEPES

Re: [ccp4bb] His tag does not bind.

2007-10-11 Thread Chun Luo
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

Re: [ccp4bb] His tag does not bind.

2007-10-11 Thread Jacob Keller
Recently, a make-or-break difference for me was making sure the pH was what I thought it was: In the presence of 50mM HEPES pH 7.0 (at room temp), the pH of my protein sample was actually nearer to 6 at 4degC, as judged by pH paper. I am not sure whether the pH difference was mainly because of

Re: [ccp4bb] His tag does not bind.

2007-10-11 Thread hong yu
150Kd is a fairly large protein for a MBP (55Kd) fusion. Six His-tag may not strong enough for it to bind to the Ni-column. If protein overexpression and solubility is not an issue, I would avoid using MBP fusion. Depend on how your protein fold, sometimes N-terminal His-tag works, sometimes C-term

Re: [ccp4bb] His tag does not bind.

2007-10-11 Thread Kendall Nettles
We have found that our His-MBP fusion doesn¹t bind well after we cut off the protein of interest, and are trying to remove it. We have to use very low salt, cold temp, and slow loading rates. You might also try batch instead of column loading. We have also had good luck adding 1-2M urea to uncut Hi

Re: [ccp4bb] His tag does not bind.

2007-10-10 Thread Tim Gruene
ednesday, 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 dete

Re: [ccp4bb] His tag does not bind.

2007-10-10 Thread Artem Evdokimov
Artem _ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of changrui lu Sent: Wednesday, October 10, 2007 9:13 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 pma

Re: [ccp4bb] His tag does not bind.

2007-10-10 Thread Das, Debanu
ds, Debanu. From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] 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 constr

[ccp4bb] His tag does not bind.

2007-10-10 Thread changrui lu
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-co