Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 62)

Vertical Streaking (contd.)
Problem Cause Solution
Twin vertical spots Improper placement of the IPG Make sure that the focused IPG strip is
or vertical doublets strip onto the gel in full contact with the gel
Temperature gradient in the gel Lower the power settings for the second-
dimension SDS-PAGE run, especially when
using cells that provide only one-sided
cooling of the gel
Use a better circulation system to improve
heat dissipation during a run
Blank vertical stripes Air bubble trapped in the agarose Ensure that the 2-D gel has a straight,
that joins the IPG strip to the top level top edge and that the IPG strip is
of the gel in direct contact with the 2-D gel along its
entire length. Squeeze out air bubbles
by pressing on the plastic backing of
the IPG strip
Use a 0.5% agarose overlay solution to
prevent the IPG strip from coming loose
or moving. To minimize the number of
bubbles in the overlay, melt the agarose
overlay solution completely prior to loading
Insufficient rehydration of a region Make sure that the IPG strip is not sticking
of the IPG strip, or tears resulting to the bottom of the rehydration tray
from improper handling, resulting
in the absence of focused protein Check the integrity of rehydrated IPG
in that region strips prior IEF
Focusing of an amphoteric Apply sample cleanup
nonprotein contaminant
(for example, phospholipid or
HEPES) prevents protein focusing
around the pI of the contaminant
Vertical Streaking (contd.)
Problem Cause Solution
Blank stripes near pH 7 Excessive DTT (>50 mM) in the Lower the amount of DTT in the
IPG sample solution rehydration solution
Blank stripes at the Salt buildup Remove ionic contaminants from the
electrodes, especially samples with Bio-Rad´s ReadyPrep 2-D
at the cathode cleanup kit or by desalting
Blank vertical regions Interfering substances; impurities Remove contaminants from the samples
in the rehydration/sample solution with the ReadyPrep 2-D cleanup kit or
by desalting
Use high-quality reagents and chemicals
for electrophoresis to minimize the risk
of impurities. Replace chemicals of
questionable or unknown shelf life, origin,
or quality, as these products can also
contribute to poor 2-D results
Air bubble trapped in the agarose Ensure that the 2-D gel has a straight,
that joins the IPG strip to the top level top edge and that the IPG strip is
of the gel in direct contact with the 2-D gel along
its entire length. Squeeze out air bubbles
by pressing on the plastic backing of
the IPG strip
Use a 0.5% agarose overlay solution to
prevent the IPG strip from coming loose or
moving. To minimize the number of bubbles
in the overlay, melt the agarose overlay
solution completely prior to loading
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2-D Electrophoresis Guide Troubleshooting