Bio-Rad Sequi-Blot™ PVDF Membrane - Manual (Page 9)

Note: Do not add acid or base to adjust pH. The
buffer will range from pH 8.1 to 8.5, depending on the
quality of Tris, glycine, dd H
2
O, and methanol.
Methanol should be analytical reagent grade, as metallic
contaminants in low grade methanol will plate on the
electrodes.
Sequi-Blot PVDF membrane stain:
0.025% Coomassie ® Blue R-250 dissolved
in a 40% MeOH solution
Sequi-Blot PVDF membrane destain:
50% MeOH solution
Section 5
Amino Acid Analysis by
Hydrolysis of Membrane
Bound Proteins
Amino acid analysis requires homogeneous
proteins. SDS-PAGE electrophoresis provides
a convenient way to purify proteins. Blotting to
Sequi-Blot PVDF membrane provides a simple
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5x working sample buffer solution:
Add 100 µl of ß-mercaptoethanol and
100 µl of bromophenol blue (BPB) solution
(0.05% w/v) to 2 ml of 5x stock sample
buffer solution.
2x working sample buffer solution:
Add 1.5 ml of distilled water, 50 µl of
ß-mercaptoethanol, and 50 µl of BPB solu-
tion (0.05% w/v) to 1 ml of 5x stock sample
buffer solution.
Towbin buffer:
25 mM Tris 3.03 g
192 M glycine 14.4 g
20% methanol 200 ml
Adjust volume to 1 liter with dd H
2
O.
Prechill the buffer before use.
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