Bio-Rad Bio-Plex® Assay Builder User Manual
Page 4
Section 3
Required and Recommended
Materials
Required Materials
The following are required for phosphoprotein detection: 1) Bio-Plex
phosphoprotein or total target assays to test for specific target proteins in
lysate samples, 2) a Bio-Plex cell lysis kit to optimally lyse cell culture or
tissue samples, and 3) a Bio-Plex phosphoprotein detection reagent kit in
order to prepare the assays and acquire data on the instrument.
*Buffers contained in this kit have not been optimized for the use with
Bio-Plex cytokine assays.
3
2
Section 2
Principle
The principle of these 96-well plate-format, bead-based assays is similar
to a capture sandwich immunoassay. An antibody directed against the
desired target protein is covalently coupled to internally dyed beads. The
coupled beads are allowed to react with a lysate sample containing target
protein. After a series of washes to remove unbound protein, a
biotinylated detection antibody specific for a different epitope is added to
the reaction. The result is the formation of a sandwich of antibodies
around the target protein. Streptavidin-phycoerythrin (streptavidin-PE) is
then added to bind to the biotinylated detection antibodies on the bead
surface.
Data from the reaction are then acquired using the Bio-Plex suspension
array system (or Luminex 100 system), a dual-laser, flow-based
microplate reader system. The contents of the well are drawn up into the
reader. The lasers and associated optics detect the internal fluorescence
of the individual dyed beads as well as the fluorescent signal on the bead
surface. This identifies each assay and reports the level of target protein
in the well. Intensity of fluorescence detected on the beads indicate the
relative quantity of targeted proteins. A high-speed digital processor
efficiently manages the data output, which is further analyzed and
presented as fluorescence intensity on Bio-Plex Manager
TM
Software, the
accompanying software package. If specific wells are identified for
comparison, the ratio of fluorescence intensity between those wells is
automatically calculated.
Bio-Plex phosphoprotein and total
target assays feature CST antibodies,
exclusively developed for Bio-Rad.
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