Bio-Rad PROTEAN II XL Cell - Manual (Page 36)

30
Troubleshooting Guide (continued)
Problem Cause Solution
7. Skewed or distorted bands. • Poor polymerization around
sample wells
• Salts in sample
• Uneven gel interface
• Degas stacking gel solution
thoroughly prior to casting;
increase ammonium
persulfate and TEMED
concentrations by 25%, or
add riboflavin phosphate to
5 μg/ml in addition to the
usual catalyst levels in the
stacking gel
• Remove salts by dialysis,
desalting column, etc.
• Increase reaction rate, overlay
carefully
8. Run taking unusually long
time.
• Buffers too concentrated
• Low current • Check buffer protocol, dilute if
necessary
• Increase current by 25–50%
9. Run too fast, poor resolution. • Buffer too dilute
• Current too high
• Check buffer protocol, dilute if
necessary
• Decrease current by 25–50%
10. Doublets observed where
a single protein species is
expected (SDS-PAGE).
• A portion of the protein may
have been re-oxidized during
the run or may not have been
fully reduced prior to run
• Prepare fresh sample buffer
solutions if over 30 days old;
increase b-mercaptoethanol
concentration in the sample
buffer
11. Observe fewer bands than
expected and one heavy
band at dye front.
• More than one band migrat-
ing at the dye front
• Increase % T of resolving gel*
12. Nonlinear pH gradient (at
basic end).
• Upper electrolyte depleted • Increase the concentration of
upper electrolyte to 100 mM
*Polyacrylamide gels are described by reference to two characteristics:
1. The total monomer concentration (%T)
2. The crosslinking monomer concentration (%C)
%T =
gm Acrylamide + gm Bis-Acrylamide
x 100
Total Volume
%C =
gm Bis-Acrylamide x 100
gm Acrylamide + gm Bis-Acrylamide