Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 60)

2-D Gel Evaluation (contd.)
Problem Cause Solution
No Spots or Fewer Spots
than Expected
In high molecular Sample may have undergone Include appropriate protease inhibitors
weight regions proteolysis prior to IEF and keep the sample on ice or in a cold
room during sample preparation
Insufficient equilibration Incubate IPG strips in sufficient volumes
of each equilibration buffer for up to
15 min with mild agitation
Poor entry of high molecular Use active sample loading in the focusing
weight proteins during rehydration tray or cup loading
(the pore size of the acrylamide in
the IPG strip is very small during
the early stages of rehydration)
Poor entry of high molecular Increase equilibration time (2 × 15 min)
weight proteins into the
second-dimension gel
Horizontal Streaking
Across the entire gel Protein overloading Use less sample
Perform prefractionation to enrich the
protein of interest and lower the amounts
of other abundant proteins
Use a longer IPG strip and larger gel size
to allow for a greater protein load
Proteins are not properly and Solubilize proteins completely using a
stably solubilized strong chaotropic extraction reagent.
The concentrations of urea, thiourea,
detergents, carrier ampholytes, and DTT
are also critical. Every sample type typically
requires a new sample preparation method
Allow sufficient time for full denaturation
and solubilization; for example, incubate
the sample in the solubilization solution at
room temperature for 1 hr before applying
it to the IPG strip
Horizontal Streaking (contd.)
Problem Cause Solution
Across the entire gel DNA contamination Treat the sample with a nuclease
Make sure that the nuclease is active and
that digestion is adequate; a very viscous
sample implies that nuclease treatment
has failed
Incomplete focusing Optimize the sample focusing time by
or overfocusing running a time course. For example,
run the sample on 6 IPG strips and
remove an IPG strip at each time point
(20 kV-hr, 30 kV-hr, 40 kV-hr, etc.)
Incomplete IPG strip rehydration Check the rehydration volumes and times
for the lengths of IPG strips used
Partial Incomplete IPG strip rehydration Check the rehydration volumes and
times for the lengths of IPG strips used
If the sample appears unevenly distributed,
or if areas of the IPG strip are not wetted
with sample, slide the IPG strip back and
forth several times along the length of the
channel in the focusing tray
Regional Protein overloading Use less sample
Perform prefractionation to enrich the
protein of interest and lower the relative
amounts of other abundant proteins
Use a longer IPG strip and larger gel size
to allow for a greater protein load
In the basic range Depletion of DTT in the basic Treat the sample with the ReadyPrep
of the gel range of the IPG strip reduction-alkylation kit prior to IEF
116 117
2-D Electrophoresis Guide Troubleshooting