[ccp4bb] ´ð¸´: [ccp4bb] protein degradation?

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

Previous message:
Subject: Re: protein degradation?
From: Eric Dollins d {- dot -} e {- dot -} dollins {- at -} GMAIL {- dot -} COM
Date: 2007-11-04

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From: "Randy J {- dot -} Read" rjr27 {- at -} CAM {- dot -} AC {- dot -} UK
Date: 2007-11-04

Subject: ´ð¸´: protein degradation?
From: Jiang Yu jyu {- at -} MAIL {- dot -} USTC {- dot -} EDU {- dot -} CN
Date: 2007-11-04

Hi Vijay,

It may be caused by the redox status of your proteins, which is normal in
redox related proteins, especially caused by cysteine residues.

The upper band may be the reduced form and the lower oxidized form, which
can be found in numerous papers.

Addition of oxidants or reductants in purification may solve the question
and homogenize your proteins, regardless of the result of SDS-PAGE.

First of all, check the redox status of your proteins.

Best Regards and Good Luck!

Sincerely yours,

Jiang Yu

School of Life Sciences

University of Science and Technology of China

_____

·¢¼þÈË: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] ´ú±í Vijay Kumar
·¢ËÍÊ±¼ä: 2007Äê11ÔÂ4ÈÕ 20:22
ÊÕ¼þÈË: CCP4BB@JISCMAIL.AC.UK
Ö÷Ìâ: [ccp4bb] protein degradation?

Hi,

I have been trying purify a N-ter his-tagged protein over-expressed in
E.coli. After purification (Ni-NTA or Co-Talon), I find two bands in SDS
PAGE which are very close each other (top band in the right MW and more
intense than the lower band). Western blot (for his-tag) of the gel gave
signal for both the bands. Mass spec results confirmed both protein bands
are the same. So I think it could be C-ter degradation of my protein. Also
the 2 bands exist after ion-exchange and sizing column.

I use commercially available complete protease inhibitor tablets (increasing
concentration has no effect) and sonication for lysis. I am wondering if
people have encountered the same problem and got any suggestions?

Thanks in advance.

Regards,

Vijay

CCP4bb navigation

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

Previous message:
Subject: Re: protein degradation?
From: Eric Dollins d {- dot -} e {- dot -} dollins {- at -} GMAIL {- dot -} COM
Date: 2007-11-04

Next message:
Subject: Re: tricky molecular replacement
From: "Randy J {- dot -} Read" rjr27 {- at -} CAM {- dot -} AC {- dot -} UK
Date: 2007-11-04