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

Help for CCPEM Archives


CCPEM Archives

CCPEM Archives


CCPEM@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

CCPEM Home

CCPEM Home

CCPEM  July 2018

CCPEM July 2018

Options

Subscribe or Unsubscribe

Subscribe or Unsubscribe

Log In

Log In

Get Password

Get Password

Subject:

Re: Tips for negative stain EM at high protein concentrations?

From:

Hiroshi Imai <[log in to unmask]>

Reply-To:

Hiroshi Imai <[log in to unmask]>

Date:

Fri, 13 Jul 2018 13:56:27 +0900

Content-Type:

text/plain

Parts/Attachments:

Parts/Attachments

text/plain (50 lines)

Dear Erik


We have used the  "rapid flush" method for negative stain EM.

This method requires higher concentration of proteins than the 
conventional negative stain EM method.

Please refer to Imai et al., 2015 Nature Communications 6:8179. In 
Methods, I hope that you would find the "rapid flush" method in the 
section of "Negative stain EM of dynein in the absence of MTs".

I hope that this information helps your research.


Best wishes,

Hiroshi

----------------------------------------------------------------------------------------------
Hiroshi Imai (PhD)
  
Laboratory of Cell Motility,
Department of Biological Sciences, Graduate School of Science,
Osaka University,
Machikaneyama-cho 1-1, Toyonaka, Osaka, 560-0043, Japan
  
E-mail: <[log in to unmask]>
_______________________________
  
  

On 2018/07/13 6:48, Erik Klontz wrote:
> Hi CCPEM community,
>
> I'm trying to solve the structure of a 140kDa complex (90+50) that has a relatively weak binding affinity (~2uM). I was going to start with negative stain, but I've found that at the low concentrations needed for negative stain, my complex falls apart. I've tried to crosslink it using GraFix and other methods, but have had very limited success crosslinking the complex without additional artifacts. This leaves me wanting to do negative stain at high protein concentrations (~2+ mg/ml). Does anyone know tricks for adsorbing FEWER particles to a grid so I can get away with using higher concentrations?
>
> Thanks!
> Erik
>
> ########################################################################
>
> To unsubscribe from the CCPEM list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCPEM&A=1
>

########################################################################

To unsubscribe from the CCPEM list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCPEM&A=1

Top of Message | Previous Page | Permalink

JiscMail Tools


RSS Feeds and Sharing


Advanced Options


Archives

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


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

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