Bio-Rad Mini-PROTEAN® Tetra Cell - Manual (Page 32)

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Section 7
Troubleshooting Guide
Problem Cause Solution
Smile effect – band
pattern curves
upward at both
sides of the gel
Center of the gel
running hotter than
either end
Power conditions
excessive
Buffer not mixed well or
buffer in upper chamber too
concentrated. Remake buffer,
ensuring thorough mixing,
especially when diluting 5x or 10x
stock
Decrease the power setting from
200 V to 150 V or fill lower chamber
to within 1 cm of top of short plate
Vertical streaking of
protein
Sample overloaded
Sample
precipitation
Dilute sample, selectively
remove predominant protein in
sample, or reduce the voltage
about 25% to minimize streaking
Centrifuge sample before
addition of SDS sample
buffer, or decrease %T of the gel*
The ratio of SDS to protein should
be enough to coat each protein
molecule with SDS, generally 1.4:1.
It may require more SDS for some
membrane protein samples
Lateral band
spreading
Diffusion of the
wells prior to
turning on the
current
Ionic strength of
the sample lower
than that of the gel
Minimize the time between sample
application and turning on the
power startup
Use same buffer in sample as in the
gel or the stacking gel
Skewed or distorted
band
Poor
polymerization
around wells
Salts in sample
Uneven gel
interface
Degas stacking gel solution
completely prior to casting;
increase ammonium persulfate and
TEMED concentrations by 25%, for
stacking gel or low %T, leave APS
the same and double the TEMED
concentration
Remove the salts by dialysis,
desalting, column, Micro Bio-
Spin™ columns, etc.
Descrease the polymerization rate.
Overlay gels very carefully