Bio-Rad Criterion™ TBE-Urea Precast Gels - Manual (Page 45)

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Problem Cause Solution
Artifactual bands at
60–70 kD Skin keratin contamination Clean all dishware, and wear gloves while
handling and loading gels
Filter all solutions (0.2–0.45 µm filter)
Poor resolution or fuzzy bands Sample volume too high If possible, load a more concentrated
sample in a lower sample buffer volume
Not enough running buffer in lower
chamber Add running buffer to fill line of lower buffer
chamber
Diffuse sample loading zone Load sample with a syringe or gel loading
pipet tip
Sample diffusion during staining 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 sample loading
buffers
Expired gel Use gels before expiration date printed on
the cassette
Criterion ™ TGX Stain-Free ™ and Criterion 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 using
the Gel Doc EZ imager 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 not visible Incorrect standards were used Use unstained standards; some prestained
standards are not detected by the Gel Doc
EZ imager. To monitor electrophoresis, use
a 1:1 mixture of unstained and prestained
standards
Dye front at bottom of gels
interfering 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
High background or low sensitivity
on a blot of a Criterion TGX Stain-
Free or Criterion Stain Free gel
(imaged using the Gel Doc EZ
imager) Low tryptophan content in proteins After activation and imaging, stain blot with
a total protein blot stain to detect missing
bands
Membrane not low-fluorescence PVDF Always use low-fluorescence PVDF
membranes to blot Criterion TGX Stain-Free
or Criterion Stain Free gels
PVDF membrane is dry Wet PVDF membrane briefly in methanol
and wash in water for 1 min before imaging
Instruction Manual and Application Guide