secreted protein purification strategy

[bheema]

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!

[protolder]

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

[fit2page]

I like reading at this forum