Bio-Rad Immun-Blot® Opti-4CN™ Colorimetric Kits User Manual

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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

~ 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.

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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