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Protein Homogenization - Soluble and insoluble protein (Mar/29/2005 )

Dear friend,

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 : )

Yong

-yongyk-

unsure.gif I do not quite understand your problem. If a protein is expressed in E.coli, after sonication, the protein is normally in cell lysate.

-leekaming-

dear yong,
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.


srini.

-srini-

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.

-sarah123-