Bio-Rad Immun-Blot® Opti-4CN™ Colorimetric Kits - Manual (Page 3)

Section 1
Introduction
Bio-Rad’s Biotin-Blot Protein Detection Kit is optimized for
nanogram level detection of total protein bound to nitrocellulose
or Zeta-Probe
® nylon membranes. The Biotin-Blot method, which
routinely detects 30 ng of membrane bound protein, is 10–50
times more sensitive than anionic stains such as Coomassie
® blue,
amido black, or fast green, none of which can be applied to nylon
membranes. Membranes analyzed for total protein with the
Biotin-Blot method can be conveniently compared with a dupli-
cate membrane which has been probed with antibodies or other
ligands. Antigenic proteins and immune complexes are easily cor-
related because direct comparisons of duplicate membranes elimi-
nate the problem of evaluating gels which have undergone
shrinkage and shape change.
The Biotin-Blot protein detection method is based on the high
affinity of avidin for biotin (K
D
~ 10 –15 M), 12-15 and the stable
avidin-biotin complex that results. To perform the assay, protein
is first bound to a nitrocellulose or nylon membrane by elec-
trophoretic transfer
1–10 or by passive dot blotting. 11 Primary and
secondary amine groups of proteins bound to a nitrocellulose or
Zeta-Probe nylon membrane are biotinylated with NHS-Biotin.
Following a wash to remove excess biotinylating reagent, the
membrane is incubated with avidin-HRP. The resultant avidin-
biotin complexes are visualized with the HRP substrate color
development procedure. Following color development, proteins
bound to nitrocellulose or nylon membranes can be seen as purple
bands or “dots.”
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