Membrane prep from insect cells

Rosario Díaz - González via methods%40net.bio.net (by rdiazg from ipb.csic.es)
Sun Jul 28 14:19:53 EST 2013


Let's give another twist: how can I purify a GST-tagged protein from Sf9
nuclei? I've tried to help lysis with hyperosmolar buffer in gradient (2M
NaCl, also tried 2M KCl) and sonication prior a GST-trap column. The buffer
included CHAPS as detergent, since I wanted to check enzimatic activity and I
had some concerns about other detergents more agressive. 

The protocol started with homogenization, centrifugation to pellet nuclei and
drop-by-drop NaCl gradient; sonication in ice to minimize genomic DNA cloggs,
centrifugation to eliminate precipitates and column loading. Unfortunately,
all the protein precipitated in the last step prior column loading.

Any suggestions?

Best,

Rose.

On Sat, 27 Jul 2013 18:36:51 -0500, Mike Autry wrote
> The protocol for 'solubilizing cells in low detergent to keep nuclei 
> intact' sounds useful, with efficient design.
> 
> Another method to keep insect cell nuclei intact for removal prior 
> to microsomal isolation (eg, for ER enzyme activity assay) is 
> 'nitrogen decompression' by Parr Bomb, charging cells at 600 PSI for 
> 5min using N2 gas tank, followed by release into atmospheric 
> pressure 15 c (http://www.parrinst.com/products/sample-
> preparation/cell-disruption/). This protocol shreds >95% of Sf21 
> insect cells, while leaving >95% of nuclei intact, as judged by 
> Trypan blue/hemocytometer. Good yield of highly-purified microsomes 
> (20 mg microsomal protein per 1E6 cells) , with a bonus: gas 
> expansion absorbs heat, so sample temperature decreases during homogenization.
> 
> In my experience, Sf21 cell microsomes tend to stick together when 
> washed with high salt (0.6M KCl, 3 mM MgCl2), but low salt seem to 
> decrease microsome clumpiness (10 mM NaHCO3, 0.2 mM MgCl2). Any tips 
> or advice on Sf21 microsomal clumpiness would be appreciated. For 
> further detail, here are examples of Parr homogenization with high 
> salt prep (http://www.jbc.org/content/272/25/15872.long#sec-1) and 
> Dounce homogenization with low salt prep 
> (http://www.jbc.org/content/275/52/41487.long#sec-1).
> 
> Mike Autry, Univ of Minnesota (no affiliation with Parr Co.)
> 
> -----Original Message-----
> From: methods-bounces from oat.bio.indiana.edu [mailto:methods-
> bounces from oat.bio.indiana.edu] On Behalf Of DK Sent: Friday, July 26,
>  2013 11:25 PM To: methods from magpie.bio.indiana.edu Subject: Re: 
> Membrane prep from insect cells
> 
> In article <mailman.547.1374861920.10461.methods from net.bio.net>, Imran 
> KHAN <mohammed.khan from ecu.edu.au> wrote:
> >Does anyone have any experience in homogenising insect cells? I am 
> >expressing a  membrane protein complex and will be purifying it by two 
> >step affinity  purification.
> 
> In which case purifying membranes might not be worth it.
> 
> >I have tried sonication  but i am not getting good protein  
> >concentration. I am using 5mM hepes, with 250mM sucrose, 2mM edta and 
> >protease  inhibitor tablet/ 5 ml buffer. After washing cells in ice 
> >cold pbs
> 
> Insect cells like higher osmolarity and are very mechanically 
> fragile. I wash them (very, very gently) in 50 mM HEPES, 160 mM NaCl 
> + 0.1% Pluronic F-68, pH 7.2.
> 
> >i sonicate
> > on low amplitude for 5 seconds and centrifuge at 1000 g to remove cell 
> >debri  and ultarcentrifuge at 100,000 g for 30 minutes to pellet 
> >membrane which i  then dissolve in 1% CHAPSO hepes buffer with 150mM 
> >NaCl overnight rotating end  over end at 4c then ultra centrifuge at 
> >100,000 g to remove undissolved  membrane.
> 
> Sonication breaks nuclei, creating significant problem with 
> viscosity and genomic DNA. I am not a fun of it. I like simply 
> dissolving membranes with 1% detergent (OG, DM, deoxycholate and the 
> likes) in low salt to keep nuclei intact, spin nuclei and load 
> directly onto the column (or bind in batch).
> 
> Is 1% CHAPSO really thast inefficient in dissolving membranes that 
> you must do the overnight thing? (That's definitely not good with 
> regard to potential proteolysis problems).
> 
> In any case, to answer you question on homogenization:
> 
> Resuspend cells in low salt buffer without sucrose and homogenize in 
> the cold using ten strokes tight teflon-glass homogenizer on a 
> motorized high troque drill. Works great!
> 
> DK
> 
> >Am i doing anything wrong here? The hepes buffer i discribed earlier 
> >was  originally used for hek cells. Is there something different i 
> >should be doing  for insect cells?
> >
> >Any help will be really appreciated
> >
> >Kind regards,
> >Imran
> >
> >
> >
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