Bio-Rad Sequi-Blot™ PVDF Membrane - Manual (Page 6)

Section 4
Electrophoretic Blotting
for Protein Sequencing
This protocol is based on the techniques
practiced by Dr. David Speicher in the Protein
Micro-chemistry Lab at The Wistar Institute.
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Each of the precautions recommended below
reduces the potential of amino terminus block-
ing during the gel purification and blotting
steps. Proteins and peptides larger than 10,000
daltons bind so strongly to PVDF that a
Polybrene coated glass fiber filter is not
required for optimal sequence analysis.
Elimination of the Polybrene coated filters
saves the time normally required for precycling,
and reduces Polybrene associated background
seen in the initial sequencing cycles.
Alternative protocols for electrophoresis and
blotting of proteins for sequence analysis,
including options for recovering peptides from
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with agitation (rotary shaker) or 24 hours with-
out. The incubation is carried out at 4 °C in a
siliconized glass tube. As the amount of protein
increases above 300 pmol, the percent of pro-
tein bound will decrease.
Note: The high binding capacity of Sequi-Blot PVDF
membrane makes it diffi-cult to block in immunoassays.
Use 0.5% casein, non-fat dry milk, or BSA, or Immun-
Blot PVDF membrane for protein immunoblotting appli-
cations for best results.
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