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Sf21 nucleus : so hard to break ??

baculo sf21 hi5 lysis

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#1 mgn67

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Posted 01 September 2011 - 04:13 AM

Hi,

I'm expressing a nuclear protein in insect cells and have encountered some issues when expressing it in Sf21 cells : my protein always remains in the insoluble fraction Posted Image
I checked with trypan blue and it seems that the nucleus won't break, though i don't treat them very gently : after ressuspending my pellet in lysis buffer (500mM NaCl, 20mM Tris, 10% glycerol, 0.2% NP-40), i homogeneize with a dounce homogenizer (50 times with the tight piston, the one which is supposed to break cell membranes and nucleus), and then sonicate.

The thing is, i'm expressing another nuclear protein but in Hi5 cells, and for those, this treatment is way enough to release my protein ! What's wrong with the Sf21 nucleus ?? Any suggestion ?

Thanks !
Morgan,

#2 protolder

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Posted 01 September 2011 - 10:03 PM

hola, probably the difference between both protein extractions is the own nature of both. I mean that insoluble protein is insoluble in your system and probably you have to denature and refold it with all the problems that this have. I had same problems in expression of a nuclear cohesin. Buena suerte





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