ThermoFisher Scientific E-PAGE Gels, Technical Manual

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9
Guidelines for Sample Preparation,
Continued
Total Sample
Volume
For best results, avoid loading less than 5 µL of sample in wells, and maintain a
uniform loading volume. If you do not have enough samples to load all the wells
of the gel, load an equal volume of deionized water into any empty wells.
E-PAGE ™ Gel Type Recommended Volume
E-PAGE ™ 96 6% Gel 15 µL
E-PAGE
™ 48
8% Gel 10 µL
We recommend loading 5–10 µL of deionized water into all wells of the
E-PAGE ™ Gel prior to loading samples or molecular weight markers.
Amount of Protein
Load up to 20 µg of protein per well of the E-PAGE
™
Gel. The amount of
protein required depends on the staining or western detection method used for
visualization. If you are unsure of how much protein to use, test a range of
concentrations to determine the optimal concentration for your sample.
Note: To ensure a proper LDS (lithium dodecyl sulfate from Loading Buffer 1)
to protein ratio, limit sample protein or lipid (from the sample) amount to
2 µg/µL of the final sample volume. Excess protein will cause poor resolution.
High Salt or
Detergent
Samples
Samples containing high salt or detergents result in loss of resolution on
E-PAGE ™ Gels. Dilute the samples such that the final concentration of the salt
or detergent in the sample is as described below. NT= not tested.
Detergent or Salt Final Concentration for
E-PAGE
™
48
Final Concentration for
E-PAGE

96
Triton ® X-100 <0.3% <0.5%
Tween ® 20 <0.3% <0.5%
CHAPS <0.3% NT
NP-40 <0.3% NT
RIPA <0.25X NT
SDS <2% (already in loading
buffer)
<4%
Tris <300 mM <200 mM
NaCl <300 mM <250 mM
Ammonium
sulfate
<100 mM NT
Sodium acetate <200 mM NT
EDTA <20 mM NT
MES Not recommended NT
DTT, Glycine,
Urea, Imidazole
No effect seen up to 500 mM NT