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 
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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] insect secretion recommendations?

[Artem Evdokimov]

Gentle spin followed by TFF - a cheap peristaltic pump plus $200 filter
cartridge will do the trick.

Artem

On Mon, Apr 20, 2020, 11:56 AM Gloria Borgstahl 
wrote:

> 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
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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>



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Re: [ccp4bb] insect secretion recommendations?

[Xavier Brazzolotto]

I used to do a low g centrifugation (100g for about 10min), collect the 
supernatant and filter it onto Polycap units (Whatman) 0,8µm/0,2µm (the size 
will depend on your volume) using a large peristaltic pump (Masterflex).

> Le 20 avr. 2020 à 17:56, Gloria Borgstahl  a écrit :
> 
> 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
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 
> 
Xavier Brazzolotto, PhD
xavier.brazzolo...@chemdef.fr

Département de Toxicologie et Risques Chimiques
Unité Neurotoxiques
Institut de Recherche Biomédicale des Armées
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91223 Brétigny sur Orge
France

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[ccp4bb] insect secretion recommendations?

[Gloria Borgstahl]

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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