Protein purification from Insect media - (Aug/30/2009 )

Hello everyone,

I’m trying to purify a 56kda His-tag protein from Insect media (protein secreted by transfected Drosophila cells). I tried Ni-NTA purification in native conditions and my protein went out in the flow through (tag shouldn’t be exposed). So, I tried Ni-NTA purification by adding 8M urea to the medium and with an imidazole gradient in the washes (10-40mM) and I didn’t see anything in the elution fractions (by blue coomasie staining). By Western blot with anti-His Ab, I get a specific ~50kda band (maybe it loses the signal peptide) in the flow-through and elution fractions. I’ve changed several conditions (imidazole, dialysis, new buffers) and no good results .
Here, some conditions of my protocol:
I start with 20ml of medium
Dialysis with PBS, overnight at 4C
I used Ni-NTA superflow resin (qiagen)
Buffer pH: 8 (lysis, wash and elution buffers)
Incubation of medium and resin for 2h at 4C (I’ve tried also overnight)
Elution with 500ul of 250mM imidazole
I haven’t used strong denaturing conditions because I need my protein for activity assays.

Have anybody else worked with protein purification from insect cell media? or is there another chromatography method more suitable to purify proteins from media supernatant?
I’m new in this issue and I don’t know what else to do. I’ll really appreciate all your suggestions .

Lucya

hi
I too purify protein (21kda) from the insect cell medium(sf911sfm). I dialyse the medium with tris/nacl ph8 overnight and next day bind the medium with BD talon resin. I can easliy get my proetin but he problem is that it is notpure enough.