Bio-Rad Mini-PROTEAN® TGX™ Precast Gels for 2-D Electrophoresis - Manual (Page 36)

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Problem Cause Solution
Bands are skewed or distorted;
lateral band spreading Too much salt in samples Remove salt from samples (dialysis,
precipitation, or other method)
Insufficient or wrong sample buffer Check buffer composition and dilution
Sample precipitation Selectively remove predominant proteins
Dilute sample in sample buffer
Insoluble materials (for example, cell
membranes) in samples Centrifuge samples to remove particulates
prior to sample loading
Artifactual bands at
60–70 kD Skin keratin contamination Clean all dishware; wear gloves while
handling and loading gels
Filter all solutions (0.2–0.45 µm filter)
Poor resolution or fuzzy bands Sample volume is too high If possible, load a more concentrated
sample in a lower sample buffer volume
Diffuse sample loading zone Load sample with a syringe or gel loading
pipet tip
Sample diffusion during staining with
Coomassie Fix gel with 40% methanol, 10% acetic acid
for 80 min prior to staining
Incompatible sample components Minimize salts, detergents, and solvents in
sample preparation and loading buffers
Expired gel Use gels before expiration date on cassette
Mini-PROTEAN ® TGX Stain-Free ™ Gels
Low sensitivity for proteins Low tryptophan content in proteins After activation and imaging, stain gel with
Bio-Safe
™ Coomassie or similar to detect
missing bands
Uneven sensitivity or fuzzy bands Gel was soaked in water or buffer prior
to activation and imaging If possible, activate and image gel
immediately after electrophoresis
Bands are too light or missing from
blot (membrane) Proteins transferred through membrane Use membrane with smaller pore size
Decrease transfer time
Decrease voltage
Standards are not visible Incorrect standards were used Use unstained standards; some prestained
standards are not detected by the imager.
To monitor electrophoresis, use a 1:1
mixture of unstained and prestained
standards
Dye front at bottom of gels
interferes with detection of proteins Sample constituents present in gel
interfering with imaging Dilute sample in gel running buffer prior to
loading
Activate and image gel, rinse in fixation
solution for 30 min, and repeat imaging
Signal intensity on blot is lower than
expected Trihalo compounds bound to
tryptophan residues inhibit binding of
some antibodies Blot gel without stain-free activation. If
signal intensity is restored, use another
(preferably polyclonal) antibody, if available
Sample bands are faint relative to
prestained standards Brightness of prestained standards can
limit exposure times for sample bands In Image Lab ™ software, select Faint
Bands to optimize exposure time or
manually define longer exposure
Adjust transform to optimize contrast for
fainter bands
Mini-PROTEAN Precast Gels