Bio-Rad Biotinylated Standards User Manual
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Section 1
Introduction
Bio-Rad's biotinylated standards are biotinylated proteins that can be
used for accurate molecular weight determinations of immune blotted pro-
teins.
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The biotinylated standards are visualized on blotted membranes next
to the sample proteins. The standards are detected in the course of the stan-
dard immune blot protocols that are performed to detect sample proteins.
The biotinylation does not significantly alter the molecular weights of the
standards. Because the sample proteins and the standards are on the same
membrane and exposed to the same reagents during detection, accurate
molecular weight markers are visualized alongside the sample proteins
without increasing the number of steps or length of incubations.
Avidin conjugates bind specifically to biotinylated proteins. Avidin is
available as Avidin-Alkaline Phosphatase Conjugate or Avidin-
Horseradish Peroxidase Conjugate. The avidin conjugates are detected by
using the appropriate color development reagent. Avidin-AP is detected
with BCIP and NBT. Avidin-HRP is detected with 4-chloro-1-naphthol.
The biotinylated standards are run with the sample proteins on an SDS-
polyacrylamide gel, and electrophoretically transferred to nitrocellulose
membrane. The standards are detected by adding the avidin conjugates to
the second antibody solution. The appropriate color development reagent is
used to detect the standards and sample proteins simultaneously.
The biotinylated standards are available in high, low, and broad molec-
ular weight ranges, and can easily be used in conjunction with Immun-
Blot
®
assay kits from Bio-Rad.
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