protein purification - (Apr/17/2006 )
Hi
Can someone help me? I am a beginer in protein purification.
I try to purify a kinase using 6xhistidine-tag and I get some problems. In westen blot I get a large quantity of protein in the crude extract (after induction) but I dont have to many protein after elution. Where can be the problem? Its a good ideea to perform the induction step at temperature lower than 37, let say 28? My protein is going in inclusion body and should I try to extract my protein from inclusion body?
Thanks a lot. Manuela
hi,
1-u can try to adjust IPTG concentration (reduce it if it is already 1mM)
2-temparature variation is necessary for some proteins alone not for all
3-if u find ur protien in inclusion bodies then u shud dissolve them in GuHCl and see whether u can purify?
4-if it is not there in inclusion bodies then u shud focus on washing buffer conditions
gud luk
Can someone help me? I am a beginer in protein purification.
I try to purify a kinase using 6xhistidine-tag and I get some problems. In westen blot I get a large quantity of protein in the crude extract (after induction) but I dont have to many protein after elution. Where can be the problem? Its a good ideea to perform the induction step at temperature lower than 37, let say 28? My protein is going in inclusion body and should I try to extract my protein from inclusion body?
Thanks a lot. Manuela
Thank you. I will try.
Like payeli says in addition to that u should also reduce shaking speed as well as try in some other expression host. if still ur protein is in inclusion body add some amount (2mM) 2-mercaptoethanol ( some times its work) in ur buffer.
all the best
Can someone help me? I am a beginer in protein purification.
I try to purify a kinase using 6xhistidine-tag and I get some problems. In westen blot I get a large quantity of protein in the crude extract (after induction) but I dont have to many protein after elution. Where can be the problem? Its a good ideea to perform the induction step at temperature lower than 37, let say 28? My protein is going in inclusion body and should I try to extract my protein from inclusion body?
Thanks a lot. Manuela
It seems like your protein has problems with binding to the tag. Try induction at 15-18 degrees, works great. Especially shifting, first grow at 37 degrees then shift to 15 degrees + IPTG after OD reaches 0.5.
Good luck!
do you mean that you dont have your protein in the supernatant or in the elute?? if supernatant then its solubilization problem if elute then its binding problem.....