Bio-Rad Sequi-Blot™ PVDF Membrane - Manual (Page 8)

dard CBB gel stain. Destain for 10-15
minutes, or until background is light blue,
with PVDF destain solution. Avoid acetic
acid in the stain and destain, as it might
cause blockage of the amino terminus.
Solutions
Stock sample buffer solution (5x without urea):
0.5 M sucrose 42.78 g
15% SDS 37.5 g
312.5 mM Tris 9.5 g
10 mM Na
2
EDTA 0.925 g
Make to a volume of 225 ml with distilled
water. Heat gently to get into solution and
adjust to pH 6.9 with 1 N HCl. Adjust to a final
volume of 250 ml. Store at 4 °C in 10 ml
aliquots.
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Transfer Protocol
These instructions are for use with a tank blot
apparatus such as the Trans-Blot
® cell. Blotting
in a Trans-Blot cell is preferable to semi-dry
transfers for protein sequencing because tank
blotting is more quantitative with higher bind-
ing yields. Follow the instructions provided
with your blotting cell for assembly.
1. For most proteins, use a Towbin buffer 5
with methanol (MeOH).
2. PVDF membrane should be clean and free
of wrinkles.
3. Wet the membrane following the protocol
in the membrane wetting section.
4. Make sure there are no bubbles between the
membrane and the gel.
5. After transfer, rinse the membrane three
times (5 minutes each) with distilled water.
6. Stain for 5 minutes with the PVDF CBB R-
250 membrane stain. Do not use the stan-
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