Dear Sivaraman
I worked on some protein with DNA contamination recently. Adding DNase
and increasing IMAC wash volume at the same time changed 280/260 ratio
and yield reproducible protein crystals, not high resolution yetIn
case your protein can't stand other treatment...
(Sorry Tommi, I hit
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf
Of Sivaraman Padavattan
Sent: Saturday, March 06, 2010 4:24 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Reg Protein purification
Dear All,
We are trying to purify an enzyme, which requires the co-factor NAD+
during catalysis
Hi Sivaraman,
One efficient way to remove nonspecifically associated DNA is to do
polyethyleneimine (PEI) precipitation. Protocols are available online.
Search for PEI precipitation.
Also look up this reference for RNA polymerase purification:
Burgess and Jendrisak. 1975 Biochemistry 14(2
dear Sivaraman,
As suggested by others, Streptomycin precipitation and
adding DnaseI to the lysis buffer are good options. You could also try
running the protein eluted from the Ni-Nta on a heparin column to remove
the nucleic acid contamination.
Ganesh
On Sat, 6 Mar 2010 17:53:42
+0530, Si
o:ccp...@jiscmail.ac.uk] On Behalf Of
Sivaraman Padavattan
Sent: Saturday, March 06, 2010 4:24 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Reg Protein purification
Dear All,
We are trying to purify an enzyme, which requires the co-factor NAD+ during
catalysis by affinity column (Ni-NTA). After indu
The concclusion that you have aggregates in the S75 is not valid in my
opinion. You might simply have a multimer which migrates >~70kDa
totest this hypothesis you should run a S200 or S6 to exclude this
option. What's your molecular weight of the monomer ?
Alternatively you might run a blue
Dear Sivaraman,
it might be difficult if the DNA binding is an intrinsic property of your
protein and important for the function. But you can try of course to degrade
the DNA using DNAseI and hope that the resulting small pieces and nucleotides
will not bind to your protein, I use sometimes st
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.
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-NT
Why don't you do a blast search in the human proteome to see whether there is a
human ortholog to the bacterial protein you co-purified, and consider also
whether the interaction would make any sense physiologically, of course. It
might be that you have discovered something interesting!
Jacob
I have seen this when expressing sub-domains of a larger protein.
Adding some (about 0.5% w/v) BOG (beta octylglucoside) worked well for
cases similar to what you've described.
Cheers,
Quyen
___
Quyen Hoang, Ph.D
Department of Biochemistry and Molecular Biology,
St
Tel: +91 22 25593761 (Lab)
email: ravima...@yahoo.com
ravima...@gmail.com
--- On Fri, 1/8/10, Sivaraman Padavattan wrote:
> From: Sivaraman Padavattan
> Subject: [ccp4bb] Reg. Protein purification
> To: CCP4BB@JISCMAIL.AC.UK
> Date: Friday, January 8, 2010, 7:41 AM
&g
An anion exchange purification (e.g. Q-Sepharose)
on a small (e.g. 1x5 mL) column with a 10 CV gradient of NaCl is a
pretty good polishing step, and quick. Dilute your protein
sample to <20 mM salt and apply to an anion exchange column at pH
8.0 or so, and elute with a 10 CV gradient of 0-0.5 M
How about using an anion exchanger after your NTA step ?
Jürgen
..
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-
Dear all,
I am trying to express the human protein using bacterial expression strain
(Rosetta) and purified using Ni-NTA affinity purification. The Molecular
weight of out protein is 47 kDa. In SDS-PAGE, we have seen that 27 kDa
contaminant protein co-eluted with our protein even at high concentrat
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