Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von supratim
dey
Gesendet: Sonntag, 16. Juni 2013 15:22
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] protein degradation
Hi
i have setup a crystallization of a complex formed by two different proteins of
molecular
Hi
i have setup a crystallization of a complex formed by two different
proteins of molecular weights 53kD and 13kD. During purification of this
complex there was slight degradation band of 53KD protein as observed from
SDS PAGE but it did not effect complex formation. Crystals appeared after 8
mont
tein solution used for
>> crystallization.
>>
>> Herman
>>
>> ------
>> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of
>> *John Domsic
>> *Sent:* Wednesday, January 16, 2013 2:22 PM
>> *To:* CCP4
*From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *John
> Domsic
> *Sent:* Wednesday, January 16, 2013 2:22 PM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] protein degradation in crystal
>
> Hi Lisa,
>
> Speed is definitely a big factor here. With
used for crystallization.
Herman
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On
Behalf Of John Domsic
Sent: Wednesday, January 16, 2013 2:22 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] protein degradation in
Were the crystals which run different on gels from the same drop, or
separate drops?
Yes, it is possible that the protein is being cleaved in the crystal
(self-cleavage?); but it may also be that it is being cleaved in the
mother liquor, and that crystallization is enriching one form or
anoth
Were the crystals which run different on gels from the same drop, or
separate drops?
Yes, it is possible that the protein is being cleaved in the crystal
(self-cleavage?); but it may also be that it is being cleaved in the
mother liquor, and that crystallization is enriching one form or
anoth
Hi Lisa,
Speed is definitely a big factor here. With a protein I work with I can
get large crystals in myriad conditions that only diffract to about 4-5
Ang. What I ended up doing was taking these crystals and seeding entire
screens. I found that not only would crystals appear sooner but it
rev
Dear Lisa
It is not uncommon to see breakdown products when you run crystals on
gel. Espesially if they are older crystals, sometimes you even see
higher molecular bands, these are probably due to intra molecular cross
links formed over time.
If you are worried about stability, try to increase
Hi All,
I have an 36KD protein which can be crystallize in two days. Most of the
crystals are very big. But all cystals have poor resolution,lower than 3.8
A. I picked some crystals, washed them in the mother solution and then run
SDS-PAGE. It is surprised to find that different cystals have differ
I agree with Mark, except that I wouldn't even try sonication, Triton or
freeze/thaw cycles in that case.
I'd look for emulsification (with a Homogenizer) in a cold room, but if you go
quickly and gently with the French Press (either in a cold room or by using a
cold piston) it might help. Don'
gt; PMSF once before lysising the cell.
>
> Yu Xiaodi
>
>
> Date: Wed, 15 Feb 2012 18:39:19 +0530
> From: sivasankarpu...@iisertvm.ac.in
> Subject: [ccp4bb] protein degradation
> To: CCP4BB@JISCMAIL.AC.UK
>
>
> Dear All,
>
> Ca
concentration).
One small "trick" you can try is wash the cell with the buffer containing PMSF
once before lysising the cell.
Yu Xiaodi
Date: Wed, 15 Feb 2012 18:39:19 +0530
From: sivasankarpu...@iisertvm.ac.in
Subject: [ccp4bb] protein degradation
To: CCP4BB@JISCMAIL.AC.UK
Dear All,
Can anybo
try experimenting with different, especially protease-deficient, E coli strains
to express the protein and try different methods to lyse the bacteria
(sonication, french-press, emulsification, bead-beater, mortar & pestle under
liquid nitrogen).
on the other hand, if you are lucky, you are just
Hi,
you may also check things like chemical degradation in SDS buffer as part of
the analysis. Esspecially your degradation pattern is very much constant
throughout your whole purification procedure.
Christian
Am Mittwoch 15 Februar 2012 14:09:19 schrieb Sivasankar Putta:
> Dear All,
>
> Can
Late induction for short time. Then immediately purify it cut down on any
unnecessary steps eg shorter spin all on ice or coldroom etc.
Jürgen
..
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria R
Alternatively, why not do a quick polish step using a long gradient on a 5
mL anion or cation exchange column, followed by a gel exclusion cleanup if
necessary? Once the proteases have been removed from the crude extract,
further degradation of purified protein should be minimal. This
purification
Hi,
I've not tried this on column cleavage before, but have you tried first
purifying the protein. cleaving the tag off the column and rerunning it through
the column to capture the tag and washing off the protein?
Also, with concentration. Going from a 50 mM buffer, and .3M salt, down to
near
Hi,
MBP tag:
1.there might be a TEV cleavage site in your MBP variant.
2. your protein needs salt to stay in solution
3. your protein forms aggregates with MBP
GST tag:
you probably concentrate a protease together with your protein. You need
a protease inhibitor kit to take care of different ty
Dear all
I am working on purification of 14 kd protein(pI 8.3, basic protein) that has
MBP(maltose binding protein, 45 kd,) tag, and same protein in other
vector(pGEX-KT) that has GST tag. During affinity purification in both cases I
used 300mM nacl and 50 mM tris, pH 7.5 buffer throughout the
bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Christian Biertuempfel
Sent: Monday, August 25, 2008 10:22 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] protein degradation
Hi Debajyoti,
There is another simple thing you can try: Raise your NaCl concentration
to 500 or 1000 mM in your
Hi Debajyoti,
There is another simple thing you can try: Raise your NaCl concentration
to 500 or 1000 mM in your lysis buffer. This helps to clean up your
sample further and it might inhibit proteases in your lysate.
Good luck,
christian
Debajyoti Dutta wrote:
Hi,
This is going to be an
ird chemistry involving the
>imidazole ring at high temp. no doubt).
>
>
>
>Artem
>
> _
>
> From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
>Debajyoti Dutta
>Sent: Sunday, August 24, 2008 3:21 AM
>To: CCP4BB@JISCMAIL.AC.UK
>Subject:
ta
Sent: Sunday, August 24, 2008 3:21 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] protein degradation
Hi again,
Thank you for all you have replied. I suspect the sonnication for such a bad
result. I am just wondering if I can use Histidine instead of Imidazole and
then buffer exchange t
most alone in the extracted material - you wil likely see a
>ribosomal p10 protein that's about pI 11 - and that's it. Remember that if
>you go this way, you should avoid using lysozyme for cell lysis since HEWL
>is mighty basic itself.
>
>
>
>Good luck,
>
>
&g
PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] protein degradation
Hi,
This is going to be an off topic question concerning this community. I have
a protein 6XHis tagged. When retrieved from the Ni-NTA column with imidazole
found to be degraded, appears like a deep band with other bands (touchin
Hi,
This is going to be an off topic question concerning this community. I have a
protein 6XHis tagged. When retrieved from the Ni-NTA column with imidazole
found to be degraded, appears like a deep band with other bands (touching each
other below the main band) in SDS PAGE. The protein is a
Let me correct myself, it is the imidazole buffer (not the nickel) and
boiling your protein in does the rest
I will try to find the exact reference for this phenomenon.
J.
Jeroen Mesters wrote:
Hi,
if I recall this correctly, it is the nickel that is in your sample
after elution and boiling
Hi,
if I recall this correctly, it is the nickel that is in your sample
after elution and boiling your protein in SDS sample buffer does the
rest.
So, could be the sample is fully okay!!!
J.
Tiago Botelho wrote:
> Hi,
>
> I also had a similar problem with one of my proteins... I had it clon
Hi,
I also had a similar problem with one of my proteins... I had it cloned in
two different plasmids, one with Cter His-tag and the other in the Nter.
Whenever I purified it using IMAC purification I would get the double band
(that I confirmed by MS and were the same).
I got ride of this "double
Another possibility, since you say MS looks identical and you are unable
to separate those two bands by other chromatografic means, is simple a
metal binding site in your protein. If the charge is changed in your
protein due to metal binding then the apparent molecular weight will
differ - that
us Drive
Evanston IL 60208
lab: 847.467.4049
cel: 773.608.9185
email: [EMAIL PROTECTED]
***
- Original Message -
From: <[EMAIL PROTECTED]>
To:
Sent: Sunday, November 04, 2007 4:18 PM
Subject: Re: [ccp4bb] protein degradation?
Some protease
etin board [mailto:[EMAIL PROTECTED] On Behalf Of
[EMAIL PROTECTED]
Sent: Sunday, November 04, 2007 5:18 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] protein degradation?
Some proteases are metal-dependent, and inhibitors for those aren't
Ni-column-compatible. "We" (meaning m
Some proteases are metal-dependent, and inhibitors for those aren't
Ni-column-compatible. "We" (meaning my students) found that it helps
to (1) work very quickly and (2) put EDTA into the fraction collector
tubes before eluting from the Ni column.
At 06:22 AM 11/4/2007, Vijay Kumar wrote:
H
Hi,
Ii happened to me before.
The protein was a dimer and by denaturing the purified protein with
urea, and rerunning the Ni-NTA, I could separate the 2 species. So the
dimer was a mixture of cleaved and uncleaved protein.
If I recall correctly, we then observed degradation again after
refoldi
Hope this helps,
Artem
_
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Vijay
Kumar
Sent: Sunday, November 04, 2007 7:22 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] protein degradation?
Hi,
I have been trying purify a N-ter his-tagged protein over-expressed in
E
Hi Vijay,
It is possible that the difference between the two bands is simply a
methionine ! If you are working with a low resolution mass spec, it
may not be very obvious. In most of the newly synthesised proteins
both in prokaryotes and eukaryotes, the initiator methionine is
removed by
On Nov 4, 2007, at 14:23, Eric Dollins wrote:
Are you expressing a eukaryotic protein? If so, you might want to
check for rare codons. There are a number of websites where you can
put in your coding sequence and check. I recently had this issue and
it turned out to be incomplete/stalled transla
China
_
发件人: CCP4 bulletin board [mailto:[EMAIL PROTECTED] 代表 Vijay Kumar
发送时间: 2007年11月4日 20:22
收件人: CCP4BB@JISCMAIL.AC.UK
主题: [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
Are you expressing a eukaryotic protein? If so, you might want to
check for rare codons. There are a number of websites where you can
put in your coding sequence and check. I recently had this issue and
it turned out to be incomplete/stalled translation rather than
proteolysis as I had several rar
Hi Vijay,
If it is C-terminal degradation, fusing the His-tag to the C-terminus
may help you get rid of it.
Wataru Kagawa
#
Wataru Kagawa, Ph. D.
Research Scientist
Protein Research Group
RIKEN (Physical and Chemical Research Institute)
W221, West R
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
signa
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