JiscMail Logo
Email discussion lists for the UK Education and Research communities

Help for CCP4BB Archives


CCP4BB Archives

CCP4BB Archives


CCP4BB@JISCMAIL.AC.UK


View:

Message:

[

First

|

Previous

|

Next

|

Last

]

By Topic:

[

First

|

Previous

|

Next

|

Last

]

By Author:

[

First

|

Previous

|

Next

|

Last

]

Font:

Proportional Font

LISTSERV Archives

LISTSERV Archives

CCP4BB Home

CCP4BB Home

CCP4BB  February 2010

CCP4BB February 2010

Options

Subscribe or Unsubscribe

Subscribe or Unsubscribe

Log In

Log In

Get Password

Get Password

Subject:

Re: Protein crystallizes while concentration

From:

Lari Lehtiö <[log in to unmask]>

Reply-To:

Lari Lehtiö <[log in to unmask]>

Date:

Thu, 18 Feb 2010 18:38:27 +0200

Content-Type:

text/plain

Parts/Attachments:

Parts/Attachments

text/plain (76 lines)

Dear Rajkumar,

I would use dialysis buttons.
E.g. http://hamptonresearch.com/product_detail.aspx?cid=10&sid=63&pid=111

Put your protein to the button and seal it with a piece of dialysis  
membrane. Place this to a linbro plate (easy to look with a  
microscope), fill the well with low salt buffer and seal with a cover  
slip.

To make the diffusion slower, you can put the dialysis button inside a  
dialysis tubing with some high salt buffer and place this to a bigger  
volume of low salt buffer.


~L~

______________________________________________________
Lari Lehtiö
Pharmacy, Department of Biochemistry and Pharmacy
Åbo Akademi University,
BioCity, FIN-20520 Turku
Finland
+358 2 215 4270
http://www.users.abo.fi/llehtio/
______________________________________________________


Quoting E rajakumar <[log in to unmask]>:

> Dear All
> I am Rajkumar, working on the protein which has unusual behavior   
> while concentration.
>
> When I kept protein in 15 mM Tris 7.5, 150 mM NaCl and 5 mM DTT, the  
>  solubility of the protein is decreases drastically and tend to   
> crystallize while concentration.
> Protein cannot be concentrated more than 3 mg/mL, however I noticed   
> white turbid protein if I force to concentrate >3mg/mL. When I   
> observed this white turbid solution under the microscope, I noticed   
> shower of tiny protein crystals which are needle in shape.
> I screened freshly purified protein (2.5 mg/mL) in different Hampton  
>  and Qiagen screens, strangely none of the conditions gave the   
> crystals.  I concentrated left over protein at 15oC at 3 mg/mL and   
> kept in the 4oC for 4 days again I noticed shower of crystals.
> This protein solubility is increased to ~20mg/mL when I kept in 15   
> Tris 7.5, 400 mM NaCl, 5% Glycerol and 5 mM DTT, but did not   
> crystallize while concentration and also after screening with   
> Hampton and Qiagen screens.
>
> My queries are
> 1. How do I get the crystals in the crystallization set up rather   
> than while concentration, so that I can control the diffusion and   
> finally nucleation?
> 2. Could anybody give me suggestions on seeding in this type of situation?
> 3. Any comments on reverse vapor diffusion for this type of protein   
> are most welcome. So I can keep protein in high ionic strength (~400  
>  mM NaCl)and diffuse against low Ionic strength or deionized water?   
> Or any other protocol?
> Any suggestions are well appreciated.
> Thanking you in advance
> Raj
>
> E. Rajakumara
> Postdoctoral Fellow  Strcutural Biology Program
> Memorial Sloan-Kettering Cancer Center
> New York-10021
> 001 212 639 7986 (Lab)  001 917 674 6266 (Mobile)
>
>
>       Get your new Email address!
> Grab the Email name you&#39;ve always wanted before someone else does!
> http://mail.promotions.yahoo.com/newdomains/aa/
>
>

Top of Message | Previous Page | Permalink

JiscMail Tools


RSS Feeds and Sharing


Advanced Options


Archives

March 2024
February 2024
January 2024
December 2023
November 2023
October 2023
September 2023
August 2023
July 2023
June 2023
May 2023
April 2023
March 2023
February 2023
January 2023
December 2022
November 2022
October 2022
September 2022
August 2022
July 2022
June 2022
May 2022
April 2022
March 2022
February 2022
January 2022
December 2021
November 2021
October 2021
September 2021
August 2021
July 2021
June 2021
May 2021
April 2021
March 2021
February 2021
January 2021
December 2020
November 2020
October 2020
September 2020
August 2020
July 2020
June 2020
May 2020
April 2020
March 2020
February 2020
January 2020
December 2019
November 2019
October 2019
September 2019
August 2019
July 2019
June 2019
May 2019
April 2019
March 2019
February 2019
January 2019
December 2018
November 2018
October 2018
September 2018
August 2018
July 2018
June 2018
May 2018
April 2018
March 2018
February 2018
January 2018
December 2017
November 2017
October 2017
September 2017
August 2017
July 2017
June 2017
May 2017
April 2017
March 2017
February 2017
January 2017
December 2016
November 2016
October 2016
September 2016
August 2016
July 2016
June 2016
May 2016
April 2016
March 2016
February 2016
January 2016
December 2015
November 2015
October 2015
September 2015
August 2015
July 2015
June 2015
May 2015
April 2015
March 2015
February 2015
January 2015
December 2014
November 2014
October 2014
September 2014
August 2014
July 2014
June 2014
May 2014
April 2014
March 2014
February 2014
January 2014
December 2013
November 2013
October 2013
September 2013
August 2013
July 2013
June 2013
May 2013
April 2013
March 2013
February 2013
January 2013
December 2012
November 2012
October 2012
September 2012
August 2012
July 2012
June 2012
May 2012
April 2012
March 2012
February 2012
January 2012
December 2011
November 2011
October 2011
September 2011
August 2011
July 2011
June 2011
May 2011
April 2011
March 2011
February 2011
January 2011
December 2010
November 2010
October 2010
September 2010
August 2010
July 2010
June 2010
May 2010
April 2010
March 2010
February 2010
January 2010
December 2009
November 2009
October 2009
September 2009
August 2009
July 2009
June 2009
May 2009
April 2009
March 2009
February 2009
January 2009
December 2008
November 2008
October 2008
September 2008
August 2008
July 2008
June 2008
May 2008
April 2008
March 2008
February 2008
January 2008
December 2007
November 2007
October 2007
September 2007
August 2007
July 2007
June 2007
May 2007
April 2007
March 2007
February 2007
January 2007


JiscMail is a Jisc service.

View our service policies at https://www.jiscmail.ac.uk/policyandsecurity/ and Jisc's privacy policy at https://www.jisc.ac.uk/website/privacy-notice

For help and support help@jisc.ac.uk

Secured by F-Secure Anti-Virus CataList Email List Search Powered by the LISTSERV Email List Manager