Bio-Rad Mini-PROTEAN® TGX™ Precast Gels for 2-D Electrophoresis - Manual (Page 20)

Stain-Free System
5
5.1 Introduction
Bio-Rad’s stain-free system eliminates the time-consuming staining and destaining steps required
by other protein detection methods. Mini-PROTEAN
® TGX Stain-Free™ gels include unique trihalo
compounds that allow rapid fluorescent detection of proteins with stain free-enabled imaging systems
— without staining.
The trihalo compounds in the gels react with tryptophan residues in a UV-induced reaction to produce
fluorescence, which can be easily detected (by stain free-enabled imagers) within gels or on low-
fluorescence PVDF membranes. Activation of the trihalo compounds in the gels adds 58 Da moieties
to available tryptophan residues and is required for protein visualization. Proteins that do not contain
tryptophan residues cannot be detected using this system. The sensitivity of the stain-free system is
comparable to staining with Coomassie Brilliant Blue for proteins with a tryptophan content >1.5%;
sensitivity superior to Coomassie staining is possible for proteins with a tryptophan content >3%.
Imaging systems that can be used with the stain-free system include the Gel Doc ™ EZ and ChemiDoc ™ MP
systems (with Image Lab ™ software).
Molecular weights of proteins can be estimated by a regression method using Image Lab software. The
software generates a standard curve using the molecular weight and relative mobility (R
f
) of standard
proteins (R
f
= distance migrated by the protein/distance migrated by the dye front). The standard curve
is then used to estimate the molecular weights of sample proteins.
Benefits of the stain-free system include:
n
Elimination of staining and destaining steps for faster results
n
Automated gel imaging and analysis
n
No background variability within a gel or between gels (as is often seen with standard
Coomassie staining)
n
Reduced organic waste by not requiring acetic acid and methanol for staining or destaining
n
Visualization of transferred (blotted) proteins on low fluorescence PVDF membranes
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