Re: [ccp4bb] insect secretion recommendations?

[Olson, Linda]

If your protein is His-tagged: spin down cells @~1500xg, adust pH with tris 
buffer and NaOH to ~pH 7.1 (spin again higher speed if necessary to remove more 
particulates) and pass over chemical resistant Ni-resin.

Linda Olson, PhD
Assistant Professor/
x-Ray Facility Manager
Dept. Biochemistry
Medical College of Wisconsin
8701 Watertown Plank Rd
Milwaukee, WI 53226

phone 414-955-8545
fax  414-456-6510

From: CCP4 bulletin board  on behalf of Gloria Borgstahl 

Sent: Monday, April 20, 2020 10:56 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] insect secretion recommendations?

ATTENTION: This email originated from a sender outside of MCW. Use caution when 
clicking on links or opening attachments.

Hi Friends,  We are secreting Spike Ecto domain into the media from insect 
cells for purification.  As we scale up I am wondering what is recommended for 
collecting the media from large volumes of culture.  Centrifugation?  
Filtration of some kind?  I imagine we need to be gentle to not lyse the insect 
cells.  Thank you, Gloria



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Re: [ccp4bb] E. coli BirA biotin ligase expression/purification

[Olson, Linda]

Dear Gloria,

This is the basic protocol we follow.  Hope it helps

Arnaud Gautier and Marlon J. Hinner (eds.), Site-Specifi c Protein Labeling: 
Methods and Protocols, Methods in Molecular Biology,
vol. 1266, DOI 10.1007/978-1-4939-2272-7_12, © Springer Science+Business Media 
New York 2015

Linda



From: CCP4 bulletin board  on behalf of Gloria Borgstahl 

Sent: Monday, October 21, 2019 12:22 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] E. coli BirA biotin ligase expression/purification

ATTENTION: This email originated from a sender outside of MCW. Use caution when 
clicking on links or opening attachments.

Dear friends in crystallography,
I know this seems unrelated, but it really isn't ... please forgive me.
We are trying to use the Avitag/BirA system to specifically biotinylate target 
proteins in an economical manner.  Are any of you purifying the BirA enzyme in 
your lab for biotinylation and if so can you help me figure out the right 
plasmid/purification protocol?   Please let me know.  Thank you.  Gloria



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Re: [ccp4bb] 3D stereo and pymol

[Olson, Linda]

Dear Daniel,

This was my question also.  I just purchased a new Mac book pro and see that 
Mantiz sells an eGPU compatible with AMD cards for Thunderbolt 3 MacOS.  I 
would be very interested in anyone’s experience or advice in making stereo work 
on the Mac platform.

Linda

On May 18, 2018, at 8:43 AM, Daniel M. Himmel, Ph. D. 
> wrote:

ATTENTION: This email originated from a sender outside of MCW. Use caution when 
clicking on links or opening attachments.

If I may jump in, I wonder if there are any hardware configurations
(monitor, 3D glasses, graphics cards, etc.) that allow 3D graphics
with a MacIntosh OSX platform.

-Daniel

On Wed, Jan 3, 2018 at 4:42 AM, Wim Burmeister 
> wrote:
I answer a bit late, but I repost a message on 3D graphics from Mai 2017 :

Hello,
we just wanted to share our experience in finding a configuration which allows 
to use 3D graphics under linux using Nvidia GeForce 3D glasses.
We had quite a hard time to find a configurations which works correctly.
We finally used Debian linux with a xfce desktop. Other recent desktops use a 
tiling which is not compatible with 3D graphics.
The hardware consists of

  *   a DELL Precision T5810  desktop computer with an Nvidia Quadro M4000 (8 
Gbyte memory, 4 DP) graphics card
  *   Nvidia GeForce 3D Vision 2 (NVIDIA GEF 3D VISION 2 GLASSES KIT) active 
stereo glasses
  *   a stereo connector PNY Quadro 4000 3D for the synchronization of graphics 
card and glasses
  *   an ASUS 24" LED 3D - VG248QE display
  *   a DisplayPort-DisplayPort cable
The Nvidia linux drivers from version 367.57 can handle the current version of 
the Nvidia glasses.
For an obscure reason a direct DP-DP connection between graphics card and 
display is absolutely required in order to obtain fully working stereo. If a 
DP-DVI dual link adapter is used, the stereo does not work on the top and the 
bottom part of the screen. This is true for a native DELL active adaptor or 
generic models. The exact reason remains unresolved, but the solution is to use 
a direct DP-DP connection. This limits the available choice of displays which 
require 120 Hz for 1080*1980 screen resolution and a DP input. We have been 
choosing a “Nvidia 3D ready” model.
There has been a considerate about of exchange about this problem on
https://devtalk.nvidia.com/default/topic/992892/linux/partially-working-stereoscopic-effect-with-3d-vision-under-debian-linux/
The setup comes with a price tag of about 1600 € free of taxes.
coot, pymol and chimera work straight without problems in hardware stereo mode. 
The experience is absolutely great.
Best
Wim
--

Wim Burmeister
Professeur
Institut de Biologie Structurale (IBS) CIBB
71 avenue des 
Martyrs

CS 20192
38044 Grenoble Cedex 9, FRANCE
E-mail: wim.burmeis...@ibs.fr
Tel:+33 (0) 457 42 87 41   Fax: +33 (0) 476 20 94 00
website