On Feb 4, 3:39 pm, Nicolas BAZEILLE <nicolas.bazei... from curie.u-psud.fr>
wrote:
> hi,
>> I try to purify a protein from yeast to assay some structural studies.
> unfortunatly, the yield is really poor and I want to find some ways to
> increase it. First, I change the medium and the temperature and the time of
> induction but I think that now it is optimal.
> Because my protein is a DNA binding protein, I think that I lost some
> proteins fixed on genomic DNA that are found in the pellet during the step
> of clarification just after breaking cells. Does it may be right ? Is there
> a way to prevent this interactions (high salt concentration, additives ??)
> I ask also, to know if there is a special manipulation to get a clear
> pellet during the step of cells collecting because my pellet is rapidly
> resolublize and the end of the run in spite of an optimal speed, resulting
> in the loss of some cells. Is there specials additives to get clear,
> compact and well packed pellet of cells, that I can put directly on the
> medium (YNB -URA galactose) at the end of induction.
>> Nicolas pHD student Institut Curie Paris france
Why not add some DNase to you lysis solution?? That should get rid of
most DNA. Not having worked with yeast, can you sonicate the cells?
That should also help in shearing the DNA.
Can't help you with the cell pelleting problem I'm afraid...
I agree with clement in that you should first look at total expression
but maybe you already have: is the yield poor after purification or do
you simply have low levels of expression?
Assuming the problem lies with purification:
High salt can help... in fact, the low yield may also be a consequence
of not using the right buffer conditions for your protein. Ideally
you would do some small scale lysis/purification experiments to
determine the optimum pH and concentration of salt for your protein
during lysis and subsequent steps.
Does it have cysteines? If so, adding reducing agents might help...
Low quantities of detergent (eg triton x100 or tween20, 0.01% or less)
can help as well...
