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Concentrating antibody supernatants from TC [View Printable]
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Carson O Genic
Group: Member
Posts: 152
Joined: Jun 22, 2005
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I like to ask for people's opions on the best method to concentrate tissue culture (TC) supernatants that contain antibody. My efforts are aimed at 1 to 5 liters of either hybridoma or CHO cell supernatants with murine or human IgG.
I started using a Vivaflow 50 PES with a 100k MWCO, as suggested by Vivasccience: http://www.vivascience.com/en/ultrafiltration/vivaflow_50.shtml
However, I've found that recovery has been absolutely horrible. I have more activity in the flow through than in the concentrate. We did not push the pressure beyond recommendation either.
I've also had poblems with the Vivaspin concentrators from this company, I have a lost antibody after spinning. Once it was clearly due to the plastic support holding the membranes breaking during the spin. Again, we're spinning these things at less than the recomended g-force. All in all this company is not on my recommend list.
I' ve got a pellicon tangential flow device on order with a 50,000 MWCO. Hopefully that will work, because this really should be quite simple. I've also switched to Millipore concnetrators (50 ml tubes-15ml supernatant size) and I'm having better luck with these.
I would love to hear what others are using to concentrate supernatants for subsequent protein A or G column purification.
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 Posted Jun 26, 2005, 18:19 PM |
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Sandy
Group: Member
Posts: 117
Joined: Nov 23, 2004
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| Carson O'Genic said: | I like to ask for people's opions on the best method to concentrate tissue culture (TC) supernatants that contain antibody. My efforts are aimed at 1 to 5 liters of either hybridoma or CHO cell supernatants with murine or human IgG.
I started using a Vivaflow 50 PES with a 100k MWCO, as suggested by Vivasccience: http://www.vivascience.com/en/ultrafiltration/vivaflow_50.shtml
However, I've found that recovery has been absolutely horrible. I have more activity in the flow through than in the concentrate. We did not push the pressure beyond recommendation either.
I've also had poblems with the Vivaspin concentrators from this company, I have a lost antibody after spinning. Once it was clearly due to the plastic support holding the membranes breaking during the spin. Again, we're spinning these things at less than the recomended g-force. All in all this company is not on my recommend list.
I' ve got a pellicon tangential flow device on order with a 50,000 MWCO. Hopefully that will work, because this really should be quite simple. I've also switched to Millipore concnetrators (50 ml tubes-15ml supernatant size) and I'm having better luck with these.
I would love to hear what others are using to concentrate supernatants for subsequent protein A or G column purification.
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You methods appear a bit complicated to me. The best way I used to do it was to filter the supernatant through 1.0 and then 0.2 micron filter. Then bring the pH to 7.0 and run it through a protein A column if your antibody is IgG. Use the right buffers (pH 7.0) to elute the antibody from your protein A column ( see www.sigma.com) then you can concentrate it down using Amicon centriprep 30M by centrifuging at 3000rpm for 10 minutes intervals. Amicon's centripreps are the best and you never loose your antibody. Hope it helped. |
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| Posted Jun 27, 2005, 16:41 PM |
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Carson O Genic
Group: Member
Posts: 152
Joined: Jun 22, 2005
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Thanks, but do you really pump liters of supernatant through a column? |
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| Posted Jun 28, 2005, 2:07 AM |
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zuzi
Group: Member
Posts: 1
Joined: Jul 19, 2007
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Hi...I use this method:
- concentrate tissue supernatant 10-20X (I use collodium membrane, but if you have enought money you can use Millipore's Centricon membrane filters in this step too). this is the longest step, it takes *days*!
- pass the concentrated supernatant on a protein A column of your choice
- concentrate again (in this case I use Millipre's Centricon 80)
Just a suggestion: use 30KDa cutoff, or you'll lose a great deal of your antibody in your flow through. To get rid of the albumin afterwards, I use a column (can't remember the name of it right now), but it's not present in large quantities, so sometimes I leave it to preserve the antibody.
I hope I've been useful!
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| Posted Jul 19, 2007, 12:49 PM |
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