I expressed the hepatitis A virus P1 gene. I checked the western blot by two ways and got good result in cell pellet.
Detail - Sample preparation:
First way: I used cell pellet and lysis in 2x sample buffer.
Second way: I used cell lysis buffer to lysis the cell pellet and centrifuge as manufacturer. The supernatant was then diluted in 5x sample buffer.
The SDS show strong appeared proteins in both two ways
The target protein was found in only first way (cell pellet).
So could you suggest me a method to purify this protein?
Thank you for your kind help!
This needs a little clarification:
Are these mammalian cells? Or insect cells perhaps?
I find my expressed protein in insect cells tends to locate within the nucleus. I lyse cells in lysis buffer (using Igepal) but I get improved recovery if I freeze/thaw the cells (ie freeze pellet and add lysis buffer to this) or if i disrupt the nucleus via sonication. Centrifugation at high speed will pellet all the membranous material and leave many nuclear proteins in solution.
Thank you, Natalie Stevens.
I expressed in insect Sf9 cells.
Could you suggest freezing condition (eg -80C or liquid nitrogen) and how many times?
Purify under native or denature condition?
Thank you,
It depends on your protein as to whether you require native or denaturing conditions, I use native conditions as my protein can be fragile but you may get a higher recovery from denaturing conditions.
I do one freeze-thaw cycle in liquid nitrogen and this seems to work for me. I have frozen at -20 before in a rush and I still find I get high yields this way.
I find there is always some protein of interest in the unbound fraction, but I attribute this mostly to HisTag degeneration.
Your protein might be either granulated or conglomerated or attached to the cel component I agrre with sonication treatment in buffer Addition of chaothropic agent at the optimized concentration might help as these agents will disturb the noncovalent interaction and thus might release the target protein
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