hello every one,
i have protein of size 21Kda which is getting IPTG induced and coming in soluble ...but when I am starting with 500 ml culture and pelleting it down and doing its lysis (50mM tris and 200mM NaCl) it not getting lysed at all
Maybe it is secreted.
The other explanation - maybe it is degraded during lysis. Do you use protease inhibitors?
50mM tris and 200mM NaCl will not lyse E. coli, I'd suggest sonication or freeze/thaw with or without lysozyme!
Hey you can use a sonication probe with 5:2 sec pulse for 5 minutes at 50% amplitude. I use 20 mMTris buffer pH8 and maintain the probe temperature (
Ramiro J. González-Duarte doing freeze-thaw in buffer 50mM tris and 200mM Nacl pH 8, then sonicating it on 15 amp with 5 sec pulse on and 10 sec pulse off
It's possible that the sonication is not working. Do you have access to a French press?
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