Membrane prep from insect cells

Imran khan via methods%40net.bio.net (by incredibleleo from yahoo.com)
Wed Jul 31 21:25:06 EST 2013


Thanks DK you have been most helpful. 

Could you please elaborate a little on the Dounce homogeniser? More specifically do you wash cells in PBS? and do you flash freeze cells in Liquid nitrogen and then dounce it? 

As you mentioned in your earlier email, you are correct i don't need to isolate membrane for affinity purification. I am trying to isolate membrane to be used in a cell free assay. As I mentioned i dissolve the membrane in 1% CHAPSO overnight however, i forgot to mention that i do this at 4C and turn the tube end over end. Would the protease inhibitor not be effective under these conditions? 

I must admit i dont have much experience with protein purification and any help would be much appreciated! 

Cheers, 
 
Imran Khan
University of Western Australia


________________________________
 From: DK <dk from no.email.thankstospam.net>
To: methods from magpie.bio.indiana.edu 
Sent: Monday, July 29, 2013 11:16 AM
Subject: RE: Membrane prep from insect cells
 

In article <mailman.551.1375045393.10461.methods from net.bio.net>, "=?ISO-8859-1?Q?Rosario_D=EDaz?=-=?ISO-8859-1?Q?Gonz=E1lez?="
        <rdiazg from ipb.csic.es> wrote:
>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. 

Here is how I purified couple proteins from insect cell nucleus: 

1. Lyse with 1% detergent in low salt with homogenization/douncing, 
(I used 1% Triton X-100 but other non-ionic detergents work just 
fine too), pellet nuclei at 1,000 g. 

2. Resuspend in whatever buffer the protein likes - absolutely no
need for high salt. (I used 40 mM Tris, 100 mM NaCl, pH 8.0) --> 
sonicate (the usual: brief bursts, ice water bath, etc). Add 
MgCl2 to 5 mM (that's optional but does bring down sizable 
amount of DNA; if your protein is DNA-binding though, propbably
a bad idea) , spin as hard as you can (ultracentrifuge is the best
but not absolutely required).

3. Bind to to the affinity matrix - all the usual considerations for 
the choice between column and batch format apply. 

>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.

Was it ever soluble to being with? In the usual crude fractionation,
it's hard to distingush between insolible protein in "inclusion bodies"
(which very A LOT in morphology and density in insect cells 
depending on the protein overexpressed) and truly nuclear protein.

If you know that your protein is soluble then you need to change 
buffer  that keeps it in solution (low salt?). If it is DNA-binding, 
digest DNA (DNAse does not work that well on chromatin, so
maybe Benzonase?) and add EDTA to chelate Mg2+ or Mn2+
before spiinning. 

DK



>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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