24
Gel temperature too high
Use external cooling during run or run out a
lower voltage.
Bands ‘smile’ across gel, band
pattern curves upward at both
sides of gel
Excess heating of gel; centre of
gel runs hotter than either end
Power conditions excessive
Insufficient buffer
Check buffer composition; buffer not mixed
well, or buffer in upper chamber too
concentrated.
Prepare new buffer, ensuring thoroughly
mixing, especially when diluting 5x or 10x
stock.
Do not exceed recommended running
conditions. Decrease power setting from
200V to 150V or fill lower chamber to within
1cm of top of short plate.
Fill inner and outer buffer chambers to ensure
that wells are completely covered.
Smiling or frowning bands with
gel lane
Overloaded proteins
Sample preparation/ buffer issues
Incorrect running conditions
Load less protein.
Minimize salts, detergents and solvents in
sample preparation and sample buffers.
Use correct voltage.
Skewed or distorted bands,
lateral band spreading
Excess salt in samples
Ionic strength of sample lower
than that of gel
Insufficient sample buffer or
wrong formulation
Diffusion prior to turning on
current
Diffusion during migration
through stacking gel
Uneven gel interface
Remove salts, from sample by dialysis or
desalting column prior to sample
preparation.
Use same buffer in samples as in gel.
Check buffer composition and dilution
instructions.
Minimize time between sample application
and power start-up.
Increase %T of stacking gel to 4.5% or 5%T.
Increase current by 25% during stacking.
Decrease polymerization rate.
Overlay gels carefully.
Rinse wells after removing comb to remove
residual acrylamide.
Vertical streaking
Overloaded samples
Sample precipitation
Dilute sample.
Selectively remove predominant protein in
sample (fractionate).
Reduce voltage by 25% to minimize
streaking.
Centrifuge samples to remove particulate
prior to sample loading.
Dilute sample in sample buffer.
Fuzzy or spurious artefactual
bands
Concentration of reducing
agent too low
Use 5% BME or 1% DTT.
Protein bands do not migrate
down as expected
Bands of interest may be neutral
or positively charged in buffer
used; pH of bands must be -2pH
units more negative than pH of
running buffer
Use SDS-PAGE or a different buffer system in
native PAGE or IEF.
Blotting
Poor protein transfer
Transfer apparatus assembled incorrectly and proteins moving
in the wrong direction.
•
Gel/membrane sandwich may be assembled in the wrong
order, or cassette inserted in wrong orientation. Check
polarity.
•
Air pockets not removed while assembling the blotting
