Bio-Rad Model 111 Mini IEF Cell - Manual (Page 23)

Section 7
Troubleshooting
7.1 General Troubleshooting
Problem Cause Solution
19
1. Excessive pooling
of water at the
cathode(-)
Agarose only.
2. Distortion in
gradient where
sample is applied.
3. Sample streaking.
4. pH gradient does
not cover
expected range.
a. Poor quality agarose.
b. Gel has not been sufficiently
blotted dry sufficiently
blotted dry prior to run.
a. Too much salt in sample.
b. Sample is applied too
near the anode.
c. Sample load excessive.
d. Sample precipitation.
a. Particles in sample.
b. Sample absorbed onto
applicator.
c. Precipitation at point of
application.
a. Focused too long or use of
excessive voltage.
b. Basic gels stored too
long.
c. Poor quality acrylamide
and Bis.
d. Old acrylamide and
Bis stock solutions.
a. Use Bio-Rad’s Zero - Mr Agrose
only
b. Blot all excess liquid from gel prior
to run.
a. Dialyze against ampholyte1%
glycine or water, or desalt with
Bio-Gel P-2 or P-6DG gel.
b. Apply elsewhere on plate,
minimum of 1 cm from anode.
c. Dilute sample.
d. Spin to remove insolubles or use
detergents.
a. Centrifuge sample before
application.
b. Change application method.
c. Try a different position.Use
additives (1% glycine,urea, non-
ionic detergents,amphoteric
detergents).
a. At recommended constant voltage,
proteins should be focused within
1.5 hours.
b. Use basic gels (pH>7)
immediately to prevent hydrolysis
of acrylamide to acrylic acid,
which causes
electroendosmosis.
c. Use highest quality acrylamide
and Bis to avoid polymerizing
acrylic acid into into the gel.
d. Prolonged storage of acrylamide
and Bis leads to acrylic acid
formation.