problem in protein expression after 8 months - (Dec/19/2008 )
Hi,
My insert is in pet22 vector.size of protein is approx. 60kDa. this is transformed in BL21(DE3). I am actually doing site directed mutagenesis of this enzyme. I check the over expression by runing a crude SDS gel.when I load freshly made samples to the gel no band appears, but next day same sample gave the band...... any explanation?
I also want to know that what are chances of any type of mutation in this vector regarding to BL21(DE3) and also XL10 Gold?
thansk in advance.
-samiSDM-
QUOTE (samiSDM @ Dec 19 2008, 08:25 AM)
Hi,
My insert is in pet22 vector.size of protein is approx. 60kDa. this is transformed in BL21(DE3). I am actually doing site directed mutagenesis of this enzyme. I check the over expression by runing a crude SDS gel.when I load freshly made samples to the gel no band appears, but next day same sample gave the band...... any explanation?
I also want to know that what are chances of any type of mutation in this vector regarding to BL21(DE3) and also XL10 Gold?
thansk in advance.
My insert is in pet22 vector.size of protein is approx. 60kDa. this is transformed in BL21(DE3). I am actually doing site directed mutagenesis of this enzyme. I check the over expression by runing a crude SDS gel.when I load freshly made samples to the gel no band appears, but next day same sample gave the band...... any explanation?
I also want to know that what are chances of any type of mutation in this vector regarding to BL21(DE3) and also XL10 Gold?
thansk in advance.
I have not had any trouble with mutations when using the pET system.
When you are checking expression, how are you doing it? I usually take 1mL of uninduced/induced cells, pellet, resuspend in lysis buffer, boil, and then run sup (after spinning down) and mix it again to run total. If you are just running the sup, you may not see your protein if it is insoluble.
When I don't see my protein in crude checks, I've often just gone ahead and tried to purify with success.
-Cheamps-
QUOTE (Cheamps @ Dec 22 2008, 12:16 PM)
QUOTE (samiSDM @ Dec 19 2008, 08:25 AM)
Hi,
My insert is in pet22 vector.size of protein is approx. 60kDa. this is transformed in BL21(DE3). I am actually doing site directed mutagenesis of this enzyme. I check the over expression by runing a crude SDS gel.when I load freshly made samples to the gel no band appears, but next day same sample gave the band...... any explanation?
I also want to know that what are chances of any type of mutation in this vector regarding to BL21(DE3) and also XL10 Gold?
thansk in advance.
My insert is in pet22 vector.size of protein is approx. 60kDa. this is transformed in BL21(DE3). I am actually doing site directed mutagenesis of this enzyme. I check the over expression by runing a crude SDS gel.when I load freshly made samples to the gel no band appears, but next day same sample gave the band...... any explanation?
I also want to know that what are chances of any type of mutation in this vector regarding to BL21(DE3) and also XL10 Gold?
thansk in advance.
I have not had any trouble with mutations when using the pET system.
When you are checking expression, how are you doing it? I usually take 1mL of uninduced/induced cells, pellet, resuspend in lysis buffer, boil, and then run sup (after spinning down) and mix it again to run total. If you are just running the sup, you may not see your protein if it is insoluble.
When I don't see my protein in crude checks, I've often just gone ahead and tried to purify with success.
I am also doing the same, except that i separate the supernatant and pellet.my protein is soluble and now it is giving expression. problem seems to be of the IPTG(someone used expired vials to make stocks......)
one more thing i would like to ask that when i load sup,my protein band gives shearing(like we get in case of DNA when it is degraded or not of good quality).what could be the reason for this band shearing?
-samiSDM-