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
Dear Friends,
I have been trying to purify a protein (27 kDa) which has a sumo plus 6 His-tag
at N-ter giving totaly a 45 kDa protein.
This protein does not express without sumo tag and has a poly basic tail (Arg
and Lys) at its N-ter. It does polymerize at acidic pHs.
When I tried to purify it
bject: [ccp4bb] purification of a strange protein
To: CCP4BB@JISCMAIL.AC.UK
Date: Tuesday, June 8, 2010, 2:13 AM
Dear colleagues,
I am now trying to purify a strange protein. The known function of the
protein was little, and it was speculated that the protein linked on the
surface of cell mem
Dear colleagues,
I am now trying to purify a strange protein. The known function of the
protein was little, and it was speculated that the protein linked on the
surface of cell membrane.
It was expressed with a Trx-tag at very low level of solubility.
Furthermore, the protein polymerized
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
tion 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 23:53:14 +
>>>
but it can't hurt.
Phoebe
Original message
Date: Thu, 19 Mar 2009 23:53:14 +
From: Kn Ly
Subject: [ccp4bb] purification
To: CCP4BB@JISCMAIL.AC.UK
Hello everyone,
I am expressing a 100 KDa eukaryotic membrane protein in E
coli. The protein
is fused to 6His-MBP in the N ter
09 23:53:14 +
>From: Kn Ly
>Subject: [ccp4bb] purification
>To: CCP4BB@JISCMAIL.AC.UK
>
>Hello everyone,
>
>I am expressing a 100 KDa eukaryotic membrane protein in E
coli. The protein
>is fused to 6His-MBP in the N terminus and the resulting mass
is ~ 150 KDa.
>
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
the Weasel comes to give New Year's greetings to the Chickens no good
intentions are in his mind.
-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Kn Ly
Sent: Thursday, March 19, 2009 7:53 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] purificati
@JISCMAIL.AC.UK
Subject: [ccp4bb] purification
Hello everyone,
I am expressing a 100 KDa eukaryotic membrane protein in E coli. The protein
is fused to 6His-MBP in the N terminus and the resulting mass is ~ 150 KDa.
However, the protein get severely degraded so after putting through a Ni-NTA
column, the
Hello everyone,
I am expressing a 100 KDa eukaryotic membrane protein in E coli. The protein
is fused to 6His-MBP in the N terminus and the resulting mass is ~ 150 KDa.
However, the protein get severely degraded so after putting through a Ni-NTA
column, the protein came out with a lot of contamin
ckens no good
intentions are in his mind.
_
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
protein.chemist protein.chemist
Sent: Thursday, February 26, 2009 5:58 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Purification with ligand
Hello,
I wanted to know i
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
Hello,
I wanted to know if there is a standard procedure for purification of
protein with ligand. I have never done this before so it will be nice to
get some help.
Thanks,
Mariah
--
Mariah Jones
Department of Biochemistry
University of Florida
09
Please respond to "Satheesh Kumar Palani Nathan" <[EMAIL PROTECTED]>
To
CCP4BB@JISCMAIL.AC.UK
cc
Subject
[ccp4bb] purification of phosphorylated proteins-off topic
Dear CCP4BB,
This is an off topic question for CCP4BB, I am posting on behalf of my
friend:
Hi,
I
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
Dear CCP4BB,
This is an off topic question for CCP4BB, I am posting on behalf of my friend:
Hi,
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
Dear All
Sorry for non-crystallography question. I am purifying
a protein using GST column. The gel filtration
chromatography is indicating that the protein is
aggregating at pH more than 7.0. I am planning to
purify the protein at the pH around 6.0. So, I want to
know the affinity of GST-fusion pr
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