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
Hi Sivaraman,
NAD+ has A259 peak absorbance. So you may not have that much nucleic acids
contamination.
However, it is not uncommon to have large amount of nucleic acids eluted
from Ni columns. Adding our TurboNuclease
(http://www.accelagen.com/TurboNuclease-protocol.htm) in lysis buffer ca
Whilst I agree with Leo that mM is a much more satisfying or rigorous unit
of concentration, the huge variability of solubility observed in proteins,
and even between point-mutants means we may as well use gigatonnes / firkin,
for all the difference it makes wrt comparing crystallisation trials.
Dear Dhirendra,
Your problems probably stems from trying to solve your structure using tantalum
clusters at too high resolution. You may also have high anisotropy in your data
since you get that negative eigenvalue error message in CTRUNCATE.
First, I suggest you look at the Harker sections fro
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
Dear Vimal, Jayashankar, Madravi,
Thanks for your Emails. I don't really know what the problem might be;
perhaps some of you could send me the data and the input parameters so
that I can reproduce the error locally. ARP/wARP is definitely not
resolution-limited (well, within limits of its a
Dear Leo,
here's a little gedankenexperiment: imagine cross-linking those solutions
you have, say with glutaraldehyde. Would you say this will *improve*
solubility because there are now less molecules?
Best wishes,
Sebastiaan Werten.
> Dear all --
>
> I have a very "stupid" question/remark con
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