Help! A Clogged HIC Cartridge (Bio-Rad)
ANONYMOUS.USER at Sunderland.AC.UK
Thu Jul 13 09:08:23 EST 1995
In article <3tsrev$s6b at charm.magnus.acs.ohio-state.edu>,
mhsieh at magnus.acs.ohio-state.edu (Ming-Ching Hsieh) says:
>Hi Dear Netters:
>Can anyone tell me how to recover a clogged HIC cartridge, which is an
>Econo-Pac Methyl HIC cartridge made by Bio-Rad? I'm using it to purify a
>apoplastic protein. I broght the infiltration solution to 2.4 M ammonium
>sulfate and applied it to the cartridge. The proteins were eluted with a linear
>gradient of ammonium sulfate from 2.4 M to zero. The specific enzyme came
>at the end of gradient. In the beginning, the flow rate was about 1 ml/min.
>However, after several uses, the flow rate was reduced to 1 ml/hr. I did clean
>the cartridge as suggested by the manufacturer, using 100mM NaOH, 20%
>and deionized water to wash. And I even washed it with 0.5M NaOH and 70%
>ethanol. They all didn't work. Please tell me what else I can do. Thanks a lot
it sounds like you have blocked the column frit or some things precipertated
out in the column.
if its the first you could try back flushing the column at a low flow rate say 0.5
ml/min for a couple of minutes, but take care nbot to over do this so as disturb
the column matrix.
if something ppt out then you really have got problems, you've used alkali to
wash through the column, would it stand a wash with dilute acid? and then
deionized water before trying again with the normal reckomended wash cycle.
hope this is of some help.
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