Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 61)

Horizontal Streaking (contd.)
Problem Cause Solution
Spots Incomplete IEF Optimize the sample focusing time by
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.)
Intermittent Contaminants such as salts, Use appropriate contaminant removal
ionic detergents (for example, techniques, such as treatment with
SDS), peptides, nucleic acids, the ReadyPrep 2-D cleanup kit
lipids, polysaccharides,
phenolic compounds
Vertical Streaking
Across the entire gel Leaking of the upper buffer Prior to inserting the gel(s) into the vertical
reservoir (cathode) of the vertical electrophoresis cell, wet the gaskets of the
electrophoresis unit electrophoresis chamber with water or
use a small amount of vacuum grease
Incomplete equilibration Increase equilibration time to 15 min
Old DTT and iodoacetamide Use fresh reagents for the equilibration step
preparations used in equilibration
Vertical Streaking (contd.)
Problem Cause Solution
At one end of the gel Protein aggregation or Dilute the sample to 3–5 µg/µl for
(cup loading) precipitation caused by cup loading
too much protein or sample
loading problems Perform a protein assay prior to IEF to
ensure correct protein load. The total
amount of protein that should be loaded
onto an IPG strip depends on the length
of the strip and the stain that will be used
to visualize the results
Load the sample using in-gel sample loading
Prolong the time on the initial low-voltage
steps and increase the voltage gradually
Field strength used for sample Reduce the field strength to ~10 V/cm
loading is too high IPG strip length
Poor protein solubility Increase the solubilizing strength of
2-D sample solution
Isolated streaking Improper rehydration of IPG strip 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
Point streaking Dust or other particles in the Filter gel solutions through a 0.45 μm
(handcast gels) gel solutions membrane and into a dust-free container
Vertical streaks Insufficient binding of SDS Check the SDS concentration (>1%) in the
connected to a spot to protein equilibration solution
Increase equilibration time:
equilibrate IPG strips for 2 × 15 min
Incorrect pH in resolving gel buffer; Ensure that the pH of the Tris buffer used
incorrect pH decreases mobility for gel casting is 8.8
of protein-SDS complexes and
causes vertical streaks
Buffer leakage Ensure that the upper buffer reservoir
is not leaking
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2-D Electrophoresis Guide Troubleshooting