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secreted protein purification strategy

secreted protein purification strategy - Recombinant Protein Forum

secreted protein purification strategy - Recombinant Protein Forum. Discuss purification, expression, and isolation of recombinant and cloned proteins here.


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  #1  
Old 05-19-2011, 07:43 PM
Pipette Filler
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Default secreted protein purification strategy



we are having a common problem in the lab with recombinant secreted protein expressed in insect cells (both Sf9 and Hi5).

1) protein does not bind when Ni-NTA or Talon is directly added to the clarified supernatant
2) 4L conditioned media preciptation with AMSO4 results in fluffy precipitates.
3) apparently, there is detergent in the media which is added to prevent shear stress to the cells during culturing, which affects binding to NiNTA. pHing the media to 8 crashes the detergent out - but the further clarified supernatant also shows poor to no binding.

What is the best way to purify secreted protein in media in insect cells.

Suggestions would be greatly appreciate!
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Old 05-20-2011, 06:11 AM
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Default Re: secreted protein purification strategy

Hola, yes serum free media have pluronic-f80 and some aminoacids like His which is interfering with Ni- resin, so make a test with a sample and the adequate ion exchanger in order to eliminate interfering agents . Or add NaCl to the medium untill 0.5-1M in order to avoid hydrophobic interactions, make a little test before, or if you have an large volumes ultrafiltration device concentrate an dyalize your medium to avoid interferring agents and put your protein in the adecuate buffer for Ni-resin. Buena suerte

Last edited by protolder; 05-20-2011 at 06:12 AM.
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Old 07-28-2012, 05:02 AM
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Default Re: secreted protein purification strategy

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