Am 24.06.2007, 12:35 Uhr, schrieb Catherine Wong <catwfy from yahoo.com>:
> I don't see a peak at all.
> what is wrong???
> then : i clean the column with 0.5M Naoh
> and GF buffer
> BUT>>>> after a volume of GF buffer pass through... the buffer can't
> seem to pass through .. i tried and waited.
> is it because of the 0.5M naoh??
> should i wash with NACL instead??
That all largely depends on the protein you want to separate. For
extracellular proteins I'd use something like 100 mM NaCl, 50 mM Tris. For
intracellular proteins a potassium-based solution is better, possibly with
some Mg too (but which may also activate proteolysis). Also note that the
cytosolic environment is reducing, some DTT or bME may help.
The first thing to make sure is that your column is properly packed.
Simply inject a sample of Dextran blue and 1 mM ATP, which should give
nice peaks at the excluded and total volume respectively, symetrical and
not too wide. The dextran blue you can see marching down the column, the
band should be sharp, without tatters and horizontal.
The next step is to calibrate the column with a couple of marker proteins,
suitable kits are commercially available from the usual suspects.
It should not normally be necessary to sanitise the column, if you filter
your samples through a 0.2 um low-protein binding membrane. A brief spin
in an Eppendorf-centrifuge can achieve the same, but be careful not to
disturb the pellet.