[Protein-analysis] Is there a way to enhance yield of soluble
DNA-binding protein in limiting DNA-protein interactions ?
(by clement from bio.mls.eng.osaka-u.ac.jp)
Mon Feb 4 20:22:26 EST 2008
Do SDS-PAGE(s) to see whether your protein is expressed well and to know
where it is. It's that simple.
In your protein sequence, did you include the signal sequence for import
into the nucleus? If you did not, you should not expect import. What kind of
lysis method you used? In many cases, DNA-protein interaction is stable
under high salt. However, it depends on what kind of interaction your
protein and DNA has.
Yeast cells should stay in the pellet nicely. If yours is easily
solubilized, perhaps your centrifuge speed is too low OR something happened
by overexpressing your protein... (which should be interesting... I don't
know. I'm not a yeast expert).
Clement Angkawidjaja, Ph.D
Laboratory of Molecular Biotechnology (Kanaya Laboratory)
Division of Advanced Science and Biotechnology
Graduate School of Engineering, Osaka University
2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
Tel./ Fax: +81-6-6879-4580
> Date: Mon, 04 Feb 2008 16:39:41 +0100
> From: Nicolas BAZEILLE <nicolas.bazeille from curie.u-psud.fr>
> Subject: [Protein-analysis] Is there a way to enhance yield of soluble
> DNA-binding protein in limiting DNA-protein interactions ?
> To: proteins from magpie.bio.indiana.edu
> Message-ID: <126.96.36.199.2.20080204140334.01b11990 from pop.curie.u-psud.fr>
> Content-Type: text/plain; charset="us-ascii"; format=flowed
> 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
> 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
> 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
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