The polybasic head indicative of your protein to have some possible
membrane association? Try a detergent in your buffers might help. Also some
of the polybasic head for protein need lipid association to be happy (PI
and PCh)?
Padayatti
On Fri, Aug 23, 2013 at 6:27 PM, Jahan Alikhajeh wrote:
>
i am not sure this would work as his protein seems to be degraded by the n
end degradation pathway. i feel like it almost needs to be expressed as a
fusion protein with some stabilizing sequence
On Fri, Aug 23, 2013 at 10:08 PM, Roger Rowlett wrote:
> Why not adopt a classical purification strat
Why not adopt a classical purification strategy? IEX-HIC-GEC. His-tag not
required. With your protein strongly basic, anion exchange seems like a
likely first step. After IEX, hydrophobic interaction or salt precipitation
followed by gel exclusion is normally enough for well-expressed proteins.
Ro
Oliver,
first, what is the source organism of the protein, human, dog, cat, insect, or
bacterial?
second, how big is the native protein, and how big are the tags you've added?
third, how many transmembrane spans are predicted (use any of the million
programs available)
fourth, when you seperate t
Hello everyone,
Just want to say thanks for your great ideas and time to reply my question.
Hope I will solve my problem soon
Kien
You can try using affinity tags on both the N- and C-termini of the
protein, eg. MBP on N and His on C.
ho
> Date: Thu, 19 Mar 2009 23:53:14 +
> From: Kn Ly
> Subject: purification
>
> Hello everyone,
>
> I am expressing a 100 KDa eukaryotic membrane protein in E coli. The prot=
> ein
Hi Kien,
As Artem pointed out earlier. Are you sure that your protein folded
correctly. You want to make sure that the protein is expressed in lipid
membrane not in inclusion body. If so, considering changing the host might
be a good idea.
Also, did you use any kind of detergent when you extract
Dear Kien,
you might also try a different resin/ metal ion. If I remember correctly,
the technician where I did my PhD had much better results with a Talon
resin using Co instead of Ni: you can use much less Imidazole, only 5-10mM
for washing and 50mM for elution.
If you can go back to cloni
Try running the Ni column as fast as possible and putting
concentrated EDTA in the fraction collector tubes before you
start, to minimize opportunities for metal-dependent proteases.
It may not be a magic bullet but it can't hurt.
Phoebe
Original message
>Date: Thu, 19 Mar 2009 23:53:1
Hi Kien-
Are you basing extensive proteolysis (degradation) on an SDS-PAGE result
alone? Are you monitoring the elution profile from Ni/NTA? Do you see
numerous A280 peaks for your elution? Sample prep of membrane proteins for
SDS-PAGE is very trial-and-error. Heating the samples may cause weird
ag
Hi,
The details of the experiment are a bit sketchy. Is this a transmembrane
protein or an associated one? What percentage of the structure is expected
to associate with the lipid bilayer? Do you know the correct orientation of
the protein with respect to the bilayer (i.e. what's inside and what's
Put the His-tag at C-terminus.
Remove rare codons
Optimize sequence for translation
You probably got truncations not degradations.
Chun
-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Kn Ly
Sent: Thursday, March 19, 2009 4:53 PM
To: CCP4BB@JISCMAI
Hi,
I wouldn't call this a standard procedure - generally speaking the most
important two parameters you have to consider are: the protein/ligand
affinity and the supply of the ligand. For tightly bound ligands, you may be
able to just add the ligand once (i.e. during cell growth and expression
Hi Mariah,
You may need to specify what type of ligand (is it a nucleotide, a
small synthetic molecule, a peptide etc ?) and also what is the affinity
between your ligand and your protein.
I have purified several protein-ligand complexes, you can go several routes.
If you have a high affinit
We have successfully used ion exchange with a very shallow gradient to
separate phosphorylated & non-phosphorylated kinases.
WRT the question on using commercially available kinases to phosphorylate
the protein, this can be used in conjunction with ion exchange
purification or SEC. It can be
I am trying to find a method to purify/separate phosphorylated protein
from unpshosphorylated proteins. I have two approach in my mind
* ion-exchange
* Fe columns, I tried this too but limited success.
Gadolinuim-charged IDA with a shallow gradient of imidazole is supposed to
separate ph
Hi there Satheesh,
In:
Activation Mechanism of the MAP Kinase ERK2 by Dual Phosphorylation .
Cell , Volume 90 , Issue 5 , Pages 859 - 869
B . Canagarajah , A . Khokhlatchev , M . Cobb , E . Goldsmith
(PDB # 2ERK)
the structure of phosphorylated ERK II was solved by co-expressing a
suitable, cons
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