Protein Homogenization - Soluble and insoluble protein (Mar/29/2005 )
Again, this is regarding to my cloning work. I clone my protein in pQE-30 vector and tranfect into E.Coli (Qiagen product). After induced by IPTG, the bacteria was harvested and protein of interest was purified using Ni-NTA method acroding to manufacturer instruction.
However when I run SDS-PAGE, I still can see a lot of my protein in the cell lysate. My protein is a soluble protein, why it can be mixed with cell lysate??
From MERCK website I got this information that: "soluble protein has to be span at 10,000g at least 30 min before it can be seperated from insoluble protein/homogenized in supernatant."
Is that true?
I will be glad if someone can comman on this
Thank everybody : )
I do not quite understand your problem. If a protein is expressed in E.coli, after sonication, the protein is normally in cell lysate.
i'm not sure but try this, it may workout.the protein expressed in e.coli when homogenized is present with the cell lysate.to separate the protein i think its better to add the mixture cell lysate+desired protein to a solution in which the only protein is soluble.this can be done by vortexing.now the supernatant(containing desired protein) is taken and is subjected to 10000 g for 30 mins to get the protein pellet.this protein pellet is mixed with gel loading buffer and loaded in to the well.i think in this way u dont find cell lysate with your desired protein in the gel.
I spin my cell extract for 30 mins at 20kRPM to make sure the supernatent is separated from the pellet. It is a bit overkill but I do it to be sure it is separated. As soon as the centrifuge stops, I pour the supernatent carefully into a container, as it can start to mix if it is left even a few minutes or if it is handled roughly.
Even a soluble protein can be partly expressed in insoluble inclusion bodies, my protein is only about 50% soluble but it is enough to purify. You can optimise soluble expression by decreased IPTG or lowering temperature.