Hello everyone. I am fairly new in protein chemistry field. I am trying to perform a protein induction hoping to purify my protein of interest using FPLC method. However, I am having trouble on not having an overexpression bands of my protein of interest when I run the crude wet cell analysis in SDS-PAGE. I can`t proceed to purification part unless I have a good overexpression for my protein of interest.
Any help or advice would be appreciated.
Thanks,
Khurshed
If your protein has a hexahistidine tag on it, you can purify it with on a column with Ni-NTA beads. Everything else washes out and, and your protein gets eluted with Imidizole.
See how faint the specific band is on my protein in the lysate. See how much I eluted with 150 mM Imidizole! That is not how much you would predicted I could elute, right? From about 6 Liters of LB with protein expressing cells induced with 250 µM IPTG, I got that ginormous band of protein, about 40-60 milligrams worth of protein. Yeah, there were obviously some impurities, but I took those out with size exclusion chromatography. The smaller band on the bottom is because my protein has a tag that is cleaved by a special protease later in the purification, but for some reason one of the E.coli proteases has that cleavage skill.
If you express a lot of protein, and it is not perfect. You can purify a lot more than you expect if you do affinity chromatography well, thoroughly, and fast:
Also some proteins just don't express well. The ribosome just doesn't feel like translating that particular sequence or doesn't fold up well, or the protein is just toxic to your cell because that is the way it is.
Khurshed Akabirov Induction for expression of protein usually refer to recombinant protein. I am not sure what kind of expression system you are using (bacteria, yeast or mammalian cells). Things you should look for is optimization the expression of your protein by different culture conditions (nutrition, temperature, induction concentration).
Depending upon whats your expression vector, youll have to play with induction conditions like amount of inducing chemical (iptg/arabinose/etc) and also the temperature and time of induction.
If your protein is a membrane protein, then go for membrane prep to visulaise your protein, as membrane proteins arnt visible to a greater extent when you are doing SDS of whole cell lysate.
You can also go for purification coloums according to the tag thats on your protein, but then it again depends upon on the expression of your protein.
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