Bio-Rad PROTEAN II XL Cell - Manual (Page 35)

29
Section 12
Troubleshooting Guide – PAGE, SDS-PAGE, 2-D IEF/SDS-PAGE
Problem Cause Solution
1. “Smile” effect — band pattern
curves upward at both sides
of the gel.
• Center of the gel running
hotter than either side
• Power conditions excessive
• Fill inner core with coolant
• Circulate coolant at
10-15 °C
• Decrease power setting
2. Diffuse tracking dye. • Decomposition of sample
solution and/or buffer stock
solutions
• Diffusion
• Prepare fresh reagents —
maximum shelf life of aque-
ous solutions is 30 days at
4 °C for buffer and monomer
stocks
• If protein bands are diffuse as
well as to the tracking dye,
increase current by 25–50%
and/or increase % T of
resolving gel*
3. Vertical streaking of protein. • Sample overload
• Sample precipitation • Dilute sample, selectively
remove predominant protein
in the sample, or reduce
current by about 25% to
minimize streaking
• Centrifuge sample or de-
crease % T of resolving gel*
4. Horizontal streaking (2-D
gels).
• Incomplete solubilization prior
to first dimension
• Interfering nucleic acids in
sample
• If urea/nonionic detergent is
not sufficient, use SDS as in
Ref. 15.5-2. Centrifugation
of sample may be necessary
(up to 100,000 x g for
30 minutes) to remove
undissolved particulates
• Treat sample with DNase or
RNase as in Ref. 15.5-1
5. Broad or diffuse protein
bands or spots (2-D).
• Diffusion due to slow
migration
• Chemical changes due to
ionic contaminants in urea
• Increase current by 20%
• Deionize urea
6. Lateral band spreading. • Diffusion out of the wells prior
to turning on the current
• Diffusion during migration
through the stacking gel
• Minimize the time between
sample application and
power start-up
• Increase % T of stacking gel
to 4.5% or 5% T, or increase
current by 25% during
stacking*