Expressing human protein in E.Coli
Posted 15 March 2011 - 04:42 PM
Posted 15 March 2011 - 11:06 PM
Hola,Have you compared your culture with one with the empty plasmid?, depending of the host sometimes there is a constitutive expression without induce. Have you any antibody against your protein or tag?. If you don´t see any signal in Western-blot I´ll recheck the sequence and frame. Give us more details, Buena suerte
I am having a hard time to express human protein in bacterial system. I have tried various concentration og IPTG, but it seems my protein is reluctant to be expressed. The incubation period tried out were 1h, 2h, 3h, 4h, and even overnight. I need assistance in this matter, Thanks.
Posted 16 March 2011 - 12:52 AM
Posted 16 March 2011 - 04:41 PM
Posted 17 March 2011 - 01:02 AM
Have you discriminated between soluble and insoluble fraction ...maybe your protein goes strictly into inclusion bodies.
Thanks for the prompt reply. I'm using pTriEx vector on Origami B strains (Novagen). Yes, I have compared between empty plasmid as negative, beta-galactosidase expression as positive, uninduced control and induced sample. But still not successful. Should I try on Western 'cos it's more sensitive in detection? I have my protein substrate with me.
Posted 17 March 2011 - 10:37 PM
Posted 17 March 2011 - 10:47 PM
Hola, but you have to see different patern of bands in each sol/insol fractions. Yes, you need see the band in direct SDS gel to try purify and have any significative amount. An option could be to put a bit (10ul) of the specific resin against tags let interact 10-20 min wash add loding buffer heat centrifuge and load the gel. if you isolate any band you coul think in made an scaling-up with more volume of sample and resin, but if you don´t purify anything, you have to revise the cloning again. Buena suerte
I have extracted both soluble and insoluble cytoplasmic fractions, but I don see any difference on the gel. Probably, I should try out western. But Is there any alternative to detect the expression of my protein before western? I really hope to see it on my SDS-PAGE. Please advice.
Posted 31 March 2011 - 05:59 PM
Posted 31 March 2011 - 10:13 PM
Hola,Yes with the plasmid from cloning strain you can tranfect mammalian cells or insect cells , in this last case for the homologous recombination method with Bsu36I digested baculoviral DNA from Clontech, Orbigen, Pharmingen or bacvector Novagen etc.This plasmid isn´t compatible with Invitrogen systems Bac-to-Bac, Baculo direct, and blue bac buena suerte con la expresión
Thanks chimaera. Just wondering the recombinant plasmid should be isolated from the cloning host and directly transfect into the expression host for the Novagen pTriEx system, am I right?