[ccp4bb] Bubbling crystals

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: Semet in non-auxotrophic strains
From: Jacob Keller j-keller2 {- at -} MD {- dot -} NORTHWESTERN {- dot -} EDU
Date: 2008-11-03

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Subject: Bubbling crystals
From: Jacob Keller j-keller2 {- at -} MD {- dot -} NORTHWESTERN {- dot -} EDU
Date: 2008-11-03

On a less mundane tack, I have noticed that when one of my proteins crystallized under very banal conditions (10mM NaHEPES 7.0, 1mM DDM, nothing else), tiny gas bubbles were trapped in the needle shower. I suspect CO2 from decarboxylation, but can anyone suggest other possibilities, or has anyone seen this in their crystalizations? I have never heard of this, except in thawing, x-ray damaged crystals.

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: j-keller2@northwestern.edu
*******************************************

----- Original Message -----
From: Jacob Keller
To: CCP4BB@JISCMAIL.AC.UK
Sent: Monday, November 03, 2008 1:37 PM
Subject: Re: [ccp4bb] Semet in non-auxotrophic strains

Dear Crystallographers,

I am overwhlemed with all of the responses--thank you all very much for your help. This listserve is a fantastic resource, and it is great that there is patience for all of the mundane methods questions! I will try to do my part to help out when I can.

Thanks,

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: j-keller2@northwestern.edu
*******************************************

----- Original Message -----
From: Cynthia Kinsland
To: Jacob Keller
Cc: CCP4BB@JISCMAIL.AC.UK
Sent: Monday, November 03, 2008 12:29 PM
Subject: Re: [ccp4bb] Semet in non-auxotrophic strains

You can express Se-Met protein in non-auxotrophs using metabolic inhibition (Doublie. Methods Enzymol. 276, 523-530, 1997 and Van Duyne et al. J. Mol. Biol. 229, 105-124, 1993).

The Studier auto-induction paper (Studier, Prot. Exp. Purif. 41, 207-234, 2005) describes an auto-induction Se-Met medium that gives sufficient incorporation of Se-Met for phasing in non-auxotrophs.

Good luck,

Cynthia

On Nov 3, 2008, at 12:56 PM, Jacob Keller wrote:

Dear Crystallographers,

I have been using an E coli strain called Tuner (DE3) pLacI, which is not auxotrophic for methionine, nor is there an auxotrophic analogue available. It may be that my protein can be expressed in other strains, but this one works well right now, and it is usually better to stay with what works well. Has anybody tried growing semet protein in non-auxotrophic strains, or are there tricks to using non-auxotrophs for semet protein?

Thanks,

Jacob

*******************************************
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: j-keller2@northwestern.edu
*******************************************

____________________
Cynthia Kinsland, Ph.D.
Cornell University
Protein Facility Director
607-255-8844

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: Semet in non-auxotrophic strains
From: Jacob Keller j-keller2 {- at -} MD {- dot -} NORTHWESTERN {- dot -} EDU
Date: 2008-11-03

Next message:
Subject: unsubscribe ccp4bb
From: Vatsan Raman srivats447 {- at -} GMAIL {- dot -} COM
Date: 2008-11-03