I'm asking you some help,
we are having a lot of troubles with "ISOPRIME APPARATUS" (Preparative
isoelectric membrane electrophoresis) that we never had before working in
a specific range of pH (4-9).
At the point we are we cannot understand which is the real problem and
nobody else has been able to give us the
right support.
We believe is not due to the kind of membranes we have prepared, anyway I'm
telling you which membranes with
Dr pH software we have prepared. From the receipe of LINEAR gradient range
pH 4-9 with the " istantaneous value"
(from "pH and deviation") several membranes have been prepared in a narrow
acid and basic range,
exactly the following :
acid range : membranes at pH 4.456 - 4.505 - 4.600 - 4.648 - 4.695
basic range : membranes at pH 6.699 - 6.749 - 6.798 - 6.848 - 6.897 - 6.947
- 7.094
Even if the "acid" separation has not been a good separation in terms of
separated isoforms, the values of volts,
amperes and watts worked normally (started from 120V - 20 mA - 4W). After
2 hr (about 800V) it began to decrease
until mA were zero, so we thought it was the power, we changed it and the
run started with new values (1400V -6 mA - 4W).
The run finished the day after at 1600V - 3 mA - 4 W).
The same day the acid membranes were removed and substituted (after washing
of everything !!) with basic membranes,
because we had to use the last acid fraction (> pH 4.695).
The run started at 2400 V - 2 mA - 4 W and after only 90 minutes it
decreased to 400 V - 10 mA - 4 W, 30 minutes later the
values were zero mA and zero Watt. We changed again with another power, the
values seemed to increase so decided
to leave the run go overnight. In the morning we found the power setted
with zero mA and zero Watt.
A technician told us that no electrical problems there were with the
instrument.
As we were not convinced we tried to separate a commom protein, HSA, using
3 of the basic membranes, pH 6.699 -
pH 6.749 - 7.094. It was very strange but it worked, the run was carried
out for 3 hrs after the prerunning.
Perplexed we decide to go on with a new experiment preparing new membranes
in the basic range : pH 6.699 - 6.947
7.045 - 7.392 (using the last basic fraction of the failed run).
The experiment failed again, it started during the prerun with water at
1600 V - 2 mA - 4 W, after 1 hr ampere and watt were
zero, we changed power but nothing changed.
Althogh this we tried to load the sample, hoping that something would have
been changed, but nothing different happened.
At this point we really don't know what to do to resolve this situation,
our small experience is not enough probably !!
We don't know if anybody else had this kind of troubles, anyway we need to
improve our efficiency
Any comments would be gratefully received.
Kind regards,
Carmelina Crisci,
IRCS - Immunochemistry Dept. - Rome
email : Carmelina.Crisci.IT_ARD01@serono.com