help! how to pack a sepharose column

hUnix nexus at interceptor.ksu.ksu.edu
Thu Apr 29 23:48:51 EST 1993


There's nothing really magic or particularly sophisticated about packing
a column. You need to swell the Sepharose beads in the buffer described
in the protocol for the prescribed time. You usually end up with a 50%
slurry by mixing it really good. Then you take a proper amount of the
50% suspension estimated on the basis of the column bed volume you
want to pack. [you can calculate the necessary volume from the data
in the supplier sheet, ml suspension of resin, or mg dry weight resin
vs. amount of protein to be purified, _bound_  by the active surface of
the column beads - this value is always given]. When you estimate the
necessary volume, you pick a proper column size that will hold this
volume. You put a bit of glasswool at the end-opening of the column and
poor the estimated amount of slurry over it. Perform several column
washes with the required buffers [there will be an activation step for
sure]. Wash with sample buffer and wait for the column to settle. [pack]
Don't let the column dry! You should have about 1 cm [~ 1/2" ] buffer
layer over the column bed surface when you apply the sample if the
column has the standard radius of 1 - 1.5 cm. You apply the sample
_carefully_ so that you _don't_ disturb the column bed surface.

Hope this helps.

--

Arseny Markov
Virology/Oncology
ex-KSU research fellow
-- 

-human research dept. by Unix, the god



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