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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

-manuela29-

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


QUOTE (manuela29 @ Apr 17 2006, 06:50 AM)
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

-payeli-

Thank you. I will try.

-manuela29-

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

-sarvind-

QUOTE (manuela29 @ Apr 17 2006, 04:50 PM)
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


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!

-smoochiepie79-

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.....

-Kathy-