[ccp4bb] Reg Protein purification

- Protein crystallography

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

Previous message:
Subject: Reg Protein purification
From: Sivaraman Padavattan s {- dot -} padavattan {- at -} GMAIL {- dot -} COM
Date: 2010-03-06

Next message:
Subject: Reg Protein purification
From: Christian Roth christian {- dot -} roth {- at -} BBZ {- dot -} UNI-LEIPZIG {- dot -} DE
Date: 2010-03-06

Subject: Reg Protein purification
From: Antony Oliver Antony {- dot -} Oliver {- at -} ICR {- dot -} AC {- dot -} UK
Date: 2010-03-06

Sivaraman,

Unfortunately not all proteins tolerate the high salt concentrations required to dissociated protein:DNA complexes.
In these cases we generally use either Benzonase or DNase I in the extraction buffer, or precipitate nucleic acid from the crude extract by adding protamine sulfate.

Interestingly NAD itself has a strong absorbance around 260nm - have you added this to the extraction buffer?

>Dear All,

>We are trying to purify an enzyme, which requires the co-factor NAD+ during catalysis by affinity column (Ni-NTA). After >induction, the bacterial cells were harvested and lysed with 20 mM Tris pH 7.2, 500 mM NaCl, 5% glycerol, 5 MM B-ME. The >resultant supernatant was passed through Ni-NTA and bound protein eluted with increasing concentration of Imidazole. The >eluted proteins was concentrated and load onto gelfiltration (Superdex S-75 16/60) column. Our protein eluted as a >aggregate along with other protein, where A260 was much greater than A280, indicative of large fraction of nucleic acid >contamination. The eluant also appeared as a smear on 1% agarose gel electrophoresis. We introduced 1M NaCl in the lysis >buffer to prevent the nucleic acid interaction. But most of our protein went in pellet after cell lysis. We look forward to your >valuable suggestion to purify the protein free of nucleic acid contamination.

>Thanks in advance,

>Sivaraman Padavattan

----
Dr Antony W. Oliver
Senior Scientist
Cancer Research UK DNA Repair Enzymes Group
Section of Structural Biology
The Institute of Cancer Research
237 Fulham Road
LONDON SW3 6JB

antony.oliver@icr.ac.uk
020 7153 5571
----

The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company Limited by Guarantee, Registered in England under Company No. 534147 with its Registered Office at 123 Old Brompton Road, London SW7 3RP.

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CCP4bb navigation

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

Previous message:
Subject: Reg Protein purification
From: Sivaraman Padavattan s {- dot -} padavattan {- at -} GMAIL {- dot -} COM
Date: 2010-03-06

Next message:
Subject: Reg Protein purification
From: Christian Roth christian {- dot -} roth {- at -} BBZ {- dot -} UNI-LEIPZIG {- dot -} DE
Date: 2010-03-06