[ccp4bb] Protein Color

- Protein crystallography

Main steps:

- Protein purification
- Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

Basic tutorials:

   - Chemistry
   - Protein
   - Peptide
   - Amino Acids

Xtal community:

- CCP4BB

CCP4bb navigation

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

Previous message:
Subject: Catalase crystals
From: Barry Silver b {- dot -} r {- dot -} silver {- at -} WARWICK {- dot -} AC {- dot -} UK
Date: 2008-09-07

Next message:
Subject: Re: Ratio of Number of Reflections to Number of restrained Parameters
From: Rajan Pillai gene1267 {- at -} GMAIL {- dot -} COM
Date: 2008-09-08

Subject: Protein Color
From: Matthew Alan Bratkowski mab259 {- at -} CORNELL {- dot -} EDU
Date: 2008-09-08

Thanks for all of the helpful suggestions. In reply to some of the
comments, I did purify by eluting with imidazole from a Ni-NTA column.
The imidazole I used is 99% pure, although I'm not sure whether it is
reagent or molecular biology grade. I also do use DTT in my lysis and
elution buffers, so these could contribute to the color.

After Ni-NTA, I dialyzed the protein into a buffer containing 10 mM Tris,
pH. 7.0, 10% Glycerol, as BME overnight, and then purified it on a
Q-column. I then did a final dialysis in buffer that contained 2 mM DTT
and 5 mM MgCl2 (although the color appears without the MgCl2 as well),
among other reagents. I would think that the two dialysis steps and the
Q-column would remove all of the imidazole. However, I did not run the
protein on a sizing column or use EDTA in any buffers. I could try using
a sizing column, and if that does not remove the color, I could use some
of the techniques suggested to determine if any metals are bound.

Matt

CCP4bb navigation

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

Previous message:
Subject: Catalase crystals
From: Barry Silver b {- dot -} r {- dot -} silver {- at -} WARWICK {- dot -} AC {- dot -} UK
Date: 2008-09-07

Next message:
Subject: Re: Ratio of Number of Reflections to Number of restrained Parameters
From: Rajan Pillai gene1267 {- at -} GMAIL {- dot -} COM
Date: 2008-09-08