Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 58)

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Problem Cause Solution
Low or zero current, With a precast gel, the tape Remove the tape
and samples do not at the bottom of the gel cassette
migrate into the gel was not removed
Insufficient buffer in the inner Fill the inner and outer buffer chambers
or outer buffer chamber to ensure that the IPG well is
completely covered
Electrical disconnection Check the electrodes and connections
Running time slower or Incorrect running buffer Check the buffer composition and type
faster than expected concentration or type
Leaking from inner Incomplete gasket seal Wet the gasket with running buffer
buffer chamber before use
Improper assembly of the gel
■
Ensure that the top edge of the short
into the electrode/ companion plate fits under the notch at the top
assembly of the gasket
■
Ensure that the top of the short plate
touches the green gasket
Isoelectric Focusing
Problem Cause Solution
Initial low or zero current Poor contact between IPG strips Make sure that the gel side of the IPG strip
and electrodes is in contact with the electrode
For the gel-side down configuration with
the PROTEAN
® i12 ™ cell, use the IPG
strip retainers
Incomplete wetting of Wet the electrode wicks with distilled or
electrode wicks deionized H
2
O until they are damp, but not
soaking wet
Incomplete IPG strip rehydration Check the rehydration volumes and times
for the lengths of IPG strips used
No current in any lane No contact between the Make sure that:
electrode assembly and
■
The electrode assembly is properly
IPG strips seated in the focusing tray
■
The IPG strips are positioned correctly,
(for example, that the gel is in direct
contact with the electrode)
No contact between the Make sure that:
electrode assembly
■
The gold contact pin of the negative (–)
and instrument assembly is in direct contact with the
cathode bar on the instrument
■
The positive (+) assembly is completely
inserted into the anode of the instrument
Voltage does not High levels of ionic contaminants Keep salt concentrations under 40 mM;
increase beyond initial in sample solution (optimum salt if necessary, desalt the sample
low voltage steps concentration is ~10 mM, though (for example with Micro Bio-Spin ™ 6
up to 40 mM can be tolerated) columns or the ReadyPrep ™ 2-D cleanup kit)
Salt collects in electrode wicks, so
replace electrode wicks from time to time
(every 2 hr) during the initial low-voltage
steps. Several hours may be needed for
ionic contaminants to leave IPG strips
Voltage does not reach Programmed voltage is too Lower the voltage maximum set for the
programmed value, high for the pH range and focusing step; the conductivity and the
or maximum voltage is length of IPG strip length and type of IPG strip determine the
reached very slowly. voltage maximum that can be reached
Note: good focusing
may be obtained even
if programmed voltage
is never reached
Ampholyte concentration is Lower the ampholyte concentration
too high. Up to 1% (v/v)
Bio-Lyte ® ampholytes may
be used, but ampholytes
increase conductivity;
therefore, voltage will be lower
with increasing concentrations
Isoelectric Focusing (contd.)
Problem Cause Solution
Excess sample during rehydration Use correct rehydration volumes for
did not enter gel, or IPG gels are the lengths of the IPG strips used
overswelled with excess sample
Voltage is too high for the IPG Program Vh for the IEF step to ensure
strip size and pH gradient complete focusing of the sample
Large fluctuations in IPG strips contain poorly Check rehydration volumes and times
voltage and current rehydrated regions, or IPG strips
have dried out during the run Make sure that the rehydration solution is
evenly distributed during rehydration and
that the IPG strips are completely covered
with mineral oil
Burning of strips Current limit is too high Use a current limit of 50 µA/IPG strip
IPG strips have dried out Make sure that the IPG strips are covered
with mineral oil or equivalent
Electrode wicks are too wet or Wet the electrode wicks with distilled
contain incorrect electrode solution or deionized H
2
O until they are damp,
not soaking wet
Incorrect rehydration Check the composition of the
solution composition rehydration solution
Sample is leaking from Cup positioning is incorrect When positioning the cup holder, make
the sample cups sure that it clicks into place at the edges
of the focusing tray
The cup is positioned in an Make sure that the IPG strips are
area of the IPG strip that is rehydrated evenly and thoroughly
not completely rehydrated
The cup is malfunctioning Replace cup
112 113
2-D Electrophoresis Guide Troubleshooting