Trouble estimating my protein size using ladder (image included)
Posted 10 December 2012 - 07:25 PM
I transformed my pET14b vector containing my target insert into Bl21(DE3) cells and induced my culture with IPTG. I ran an SDS-PAGE to check for the solubility, expression, and whether my protein got induced. The different fractions cellular fractions I ran on gel include total cell protein fraction and soluble cytoplasmic fraction.
My problem is I cannot locate my protein on the SDS-PAGE gel. My protein size is approximately 21.38 kilodaltons. I cannot really know how my inductions really went? Can anyone provide me with feedback please. I would greatly appreciate it.
My image organization
far left-ladder, total cell protein induced, total cell protein uninduced, soluble cytoplasmic induced, soluble cytoplasmic uninduced-far right.
Here is the image
For ladder image here is the link http://tools.invitro...ned_Std_man.pdf
Posted 10 December 2012 - 08:03 PM
I've attached the band I'm thinking is correct, and what I think the markers are. I can't be positive, as I usually use multicolored markers, and I usually get a feel for how the markers run on a given gel type over time, so I can tell at a glance which markers are which.
I'll also note that with many protein expressions that I do, I do not see my protein in the total extract at all, but only see it after enrichment after binding to affinity resin and eluting (for many His-tagged proteins in particular). So you may want to try a trial run of your first purification step to see if you get a more prominent band at the right size.
Posted 12 December 2012 - 11:28 AM
Ok I am waiting on my anti-his antibody and purification kit and will do as suggested. I hope for the best.