Re: [ccp4bb] Off-topic question about SEC

2017-01-12 Thread Nicolas RICHET 
Dear Reza,
Regarding the molecular weights (60 to 360 kDa), the low salt
concentration seems to drive the assembly of an hexamer.
Salt concentration could have a drastic effect on protein oligomerization.
If your A280 profile of the 360 kDa peak looks good (symmetric and in the
resolution limits), it is very likely to be an "hexamerization"!
As Nicolas FOOS said, you could confirm it using DLS or SEC-MALS (even
better).
Best wishes,
Nicolas




*Nicolas RICHET, Ph. D.Post-Doctoral ResearcherUniversity College Cork*
*School of Microbiology*

*Food Science and Technology Building*

*College Road*

*Cork City*
*IRELAND*


*Mobile: +353 (0)838385151*

2017-01-12 9:26 GMT+00:00 Nicolas FOOS <nicolas.f...@esrf.fr>:

> Dear Reza,
>
>
> in the past I had work with protein able to oligomerize reversibly but
> when oligomerization happened, even if I was able to separate and obtain
> monomeric protein,
>
> protein was not in a good condition.
>
>
> Have you try to characterize the two different states by DLS ? To
> discriminate "interaction with resin" that you suspect from oligomerization.
>
> Nicolas
>
>
> Nicolas Foos
> PhD
> Structural Biology Group
> European Synchrotron Radiation Facility (E.S.R.F)
> 71, avenue des Martyrs
> CS 40220
> 38043 GRENOBLE Cedex 9+33 (0)6 76 88 14 87 <+33%206%2076%2088%2014%2087>+33 
> (0)4 76 88 45 19 <+33%204%2076%2088%2045%2019>
>
> On 12/01/2017 01:50, Christopher Colbert wrote:
>
> What's your monomeric molecular weight?  Increased salt concentration can
> easily drive oligomerization.
>
> What is your evidence that it interacts with the resin?
>
> Cheers,
>
> Chris
>
> --
> Christopher L. Colbert, Ph.D.
> Associate Professor
> Department of Chemistry and Biochemistry
> North Dakota State University
> P.O. Box 6050 Dept. 2710
> Fargo, ND 58108-6050
> PH: (701) 231-7946
> FAX: (701) 231-8324
>
> From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Reza
> Khayat <rkha...@ccny.cuny.edu>
> Reply-To: Reza Khayat <rkha...@ccny.cuny.edu>
> Date: Wednesday, January 11, 2017 6:42 PM
> To: "CCP4BB@JISCMAIL.AC.UK" <CCP4BB@JISCMAIL.AC.UK>
> Subject: Re: [ccp4bb] Off-topic question about SEC
>
> ​All these make sense. Protein is very strange cause it goes from 60kDa
> (globular) to an apparent 360kDa. Process is reversible too.
>
>
> Reza Khayat, PhD
> Assistant Professor
> City College of New York
> Department of Chemistry
> New York, NY 10031
> ------
> *From:* CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Keller,
> Jacob <kell...@janelia.hhmi.org>
> *Sent:* Wednesday, January 11, 2017 7:39 PM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] Off-topic question about SEC
>
>
> Yes if it either
>
>
>
> A) oligomerizes
>
> B) significantly changes shape
>
> C) aggregates reversibly
>
>
>
> On option B: Lower NaCl could make the protein “appear” bigger by
> unfolding it a bit; hydrophobic interactions should be weaker in lower NaCl.
>
>
>
> JPK
>
>
>
>
>
>
>
>
>
>
>
> Artem
>
> www.harkerbio.com
>
> "where wild SEC columns roam free"
>
>
>
> On Jan 11, 2017 7:22 PM, "Reza Khayat" <rkha...@ccny.cuny.edu> wrote:
>
> Hi,
>
>
>
> Sorry for the off-topic question. Can a protein in lower [NaC] run faster
> on a SEC than at higher [NaCl] (i.e. elute at an earlier volume)? The
> protein elutes well within the resolution limits of the SEC
> with a symmetric gaussian A280 profile. I know that at lower [NaCl] the
> protein can elute later because it may interact with the matrix.  Thanks.
>
>
>
> Best wishes,
> Reza
>
>
>
> Reza Khayat, PhD
>
> Assistant Professor
>
> City College of New York
>
> Department of Chemistry
>
> New York, NY 10031
>
>
>
>
>

Re: [ccp4bb] Off-topic question about SEC

2017-01-12 Thread Nicolas FOOS 

Dear Reza,


in the past I had work with protein able to oligomerize reversibly but 
when oligomerization happened, even if I was able to separate and obtain 
monomeric protein,


protein was not in a good condition.


Have you try to characterize the two different states by DLS ? To 
discriminate "interaction with resin" that you suspect from oligomerization.


Nicolas


Nicolas Foos
PhD
Structural Biology Group
European Synchrotron Radiation Facility (E.S.R.F)
71, avenue des Martyrs
CS 40220
38043 GRENOBLE Cedex 9
+33 (0)6 76 88 14 87
+33 (0)4 76 88 45 19

On 12/01/2017 01:50, Christopher Colbert wrote:
What's your monomeric molecular weight?  Increased salt concentration 
can easily drive oligomerization.


What is your evidence that it interacts with the resin?

Cheers,

Chris

--
Christopher L. Colbert, Ph.D.
Associate Professor
Department of Chemistry and Biochemistry
North Dakota State University
P.O. Box 6050 Dept. 2710
Fargo, ND 58108-6050
PH: (701) 231-7946
FAX: (701) 231-8324

From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK 
<mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Reza Khayat 
<rkha...@ccny.cuny.edu <mailto:rkha...@ccny.cuny.edu>>
Reply-To: Reza Khayat <rkha...@ccny.cuny.edu 
<mailto:rkha...@ccny.cuny.edu>>

Date: Wednesday, January 11, 2017 6:42 PM
To: "CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>" 
<CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>>

Subject: Re: [ccp4bb] Off-topic question about SEC

​All these make sense. Protein is very strange cause it goes from 
60kDa (globular) to an apparent 360kDa. Process is reversible too.



Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

*From:* CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK 
<mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Keller, Jacob 
<kell...@janelia.hhmi.org <mailto:kell...@janelia.hhmi.org>>

*Sent:* Wednesday, January 11, 2017 7:39 PM
*To:* CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>
*Subject:* Re: [ccp4bb] Off-topic question about SEC

Yes if it either

A) oligomerizes

B) significantly changes shape

C) aggregates reversibly

On option B: Lower NaCl could make the protein “appear” bigger by 
unfolding it a bit; hydrophobic interactions should be weaker in lower 
NaCl.


JPK

Artem

www.harkerbio.com <http://www.harkerbio.com>

"where wild SEC columns roam free"

On Jan 11, 2017 7:22 PM, "Reza Khayat" <rkha...@ccny.cuny.edu 
<mailto:rkha...@ccny.cuny.edu>> wrote:


Hi,

Sorry for the off-topic question. Can a protein in lower [NaC] run
faster on a SEC than at higher [NaCl] (i.e. elute at an earlier
volume)? The protein elutes well within the resolution limits of
the SEC with a symmetric gaussian A280 profile. I know that at
lower [NaCl] the protein can elute later because it may
interact with the matrix.  Thanks.

Best wishes,
Reza

Reza Khayat, PhD

Assistant Professor

City College of New York

Department of Chemistry

New York, NY 10031

Re: [ccp4bb] Off-topic question about SEC

2017-01-11 Thread Christopher Colbert 
What's your monomeric molecular weight?  Increased salt concentration can 
easily drive oligomerization.

What is your evidence that it interacts with the resin?

Cheers,

Chris

--
Christopher L. Colbert, Ph.D.
Associate Professor
Department of Chemistry and Biochemistry
North Dakota State University
P.O. Box 6050 Dept. 2710
Fargo, ND 58108-6050
PH: (701) 231-7946
FAX: (701) 231-8324

From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Reza Khayat <rkha...@ccny.cuny.edu<mailto:rkha...@ccny.cuny.edu>>
Reply-To: Reza Khayat <rkha...@ccny.cuny.edu<mailto:rkha...@ccny.cuny.edu>>
Date: Wednesday, January 11, 2017 6:42 PM
To: "CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>" 
<CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: Re: [ccp4bb] Off-topic question about SEC


​All these make sense. Protein is very strange cause it goes from 60kDa 
(globular) to an apparent 360kDa. Process is reversible too.


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Keller, Jacob 
<kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>>
Sent: Wednesday, January 11, 2017 7:39 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] Off-topic question about SEC

Yes if it either

A) oligomerizes
B) significantly changes shape
C) aggregates reversibly

On option B: Lower NaCl could make the protein “appear” bigger by unfolding it 
a bit; hydrophobic interactions should be weaker in lower NaCl.

JPK





Artem
www.harkerbio.com<http://www.harkerbio.com>
"where wild SEC columns roam free"

On Jan 11, 2017 7:22 PM, "Reza Khayat" 
<rkha...@ccny.cuny.edu<mailto:rkha...@ccny.cuny.edu>> wrote:

Hi,



Sorry for the off-topic question. Can a protein in lower [NaC] run faster on a 
SEC than at higher [NaCl] (i.e. elute at an earlier volume)? The protein elutes 
well within the resolution limits of the SEC with a symmetric gaussian A280 
profile. I know that at lower [NaCl] the protein can elute later because it may 
interact with the matrix.  Thanks.



Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

Re: [ccp4bb] Off-topic question about SEC

2017-01-11 Thread Keller, Jacob 
Whoa! Big change! Any possible physiological relevance?

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Reza 
Khayat
Sent: Wednesday, January 11, 2017 7:43 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Off-topic question about SEC


​All these make sense. Protein is very strange cause it goes from 60kDa 
(globular) to an apparent 360kDa. Process is reversible too.


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Keller, Jacob 
<kell...@janelia.hhmi.org<mailto:kell...@janelia.hhmi.org>>
Sent: Wednesday, January 11, 2017 7:39 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] Off-topic question about SEC

Yes if it either

A) oligomerizes
B) significantly changes shape
C) aggregates reversibly

On option B: Lower NaCl could make the protein “appear” bigger by unfolding it 
a bit; hydrophobic interactions should be weaker in lower NaCl.

JPK





Artem
www.harkerbio.com<http://www.harkerbio.com>
"where wild SEC columns roam free"

On Jan 11, 2017 7:22 PM, "Reza Khayat" 
<rkha...@ccny.cuny.edu<mailto:rkha...@ccny.cuny.edu>> wrote:

Hi,



Sorry for the off-topic question. Can a protein in lower [NaC] run faster on a 
SEC than at higher [NaCl] (i.e. elute at an earlier volume)? The protein elutes 
well within the resolution limits of the SEC with a symmetric gaussian A280 
profile. I know that at lower [NaCl] the protein can elute later because it may 
interact with the matrix.  Thanks.



Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

Re: [ccp4bb] Off-topic question about SEC

2017-01-11 Thread Keller, Jacob 
Yes if it either

A) oligomerizes
B) significantly changes shape
C) aggregates reversibly

On option B: Lower NaCl could make the protein “appear” bigger by unfolding it 
a bit; hydrophobic interactions should be weaker in lower NaCl.

JPK





Artem
www.harkerbio.com
"where wild SEC columns roam free"

On Jan 11, 2017 7:22 PM, "Reza Khayat" 
> wrote:

Hi,



Sorry for the off-topic question. Can a protein in lower [NaC] run faster on a 
SEC than at higher [NaCl] (i.e. elute at an earlier volume)? The protein elutes 
well within the resolution limits of the SEC with a symmetric gaussian A280 
profile. I know that at lower [NaCl] the protein can elute later because it may 
interact with the matrix.  Thanks.



Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

Re: [ccp4bb] Off-topic question about SEC

2017-01-11 Thread Artem Evdokimov 
Yes if it either

A) oligomerizes
B) significantly changes shape
C) aggregates reversibly

Artem
www.harkerbio.com
"where wild SEC columns roam free"

On Jan 11, 2017 7:22 PM, "Reza Khayat"  wrote:

> Hi,
>
>
> Sorry for the off-topic question. Can a protein in lower [NaC] run faster
> on a SEC than at higher [NaCl] (i.e. elute at an earlier volume)? The
> protein elutes well within the resolution limits of the SEC
> with a symmetric gaussian A280 profile. I know that at lower [NaCl] the
> protein can elute later because it may interact with the matrix.  Thanks.
>
>
> Best wishes,
> Reza
>
>
> Reza Khayat, PhD
> Assistant Professor
> City College of New York
> Department of Chemistry
> New York, NY 10031
>
>
>

[ccp4bb] Off-topic question about SEC

2017-01-11 Thread Reza Khayat 
Hi,


Sorry for the off-topic question. Can a protein in lower [NaC] run faster on a 
SEC than at higher [NaCl] (i.e. elute at an earlier volume)? The protein elutes 
well within the resolution limits of the SEC with a symmetric gaussian A280 
profile. I know that at lower [NaCl] the protein can elute later because it may 
interact with the matrix.  Thanks.


Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031