CST Guide:

Section III: Workflow Tools
chapter 23: Localization & classification
Optimal antibody-to-dye ratio is critical for maximum performance.
Custom conjugations are optimized by degree of
labeling testing to identify the optimal antibody:
dye molecule ratio, resulting in conjugates with
maximum performance.
DOL 2.34
DOL 4.28
DOL 5.51
DOL 7.27
How CST conjugated antibodies can benefit your research:
• Rigorously Validated: optimal conjugation chemistry and DOL are determined for each antibody
in their intended application, producing conjugates with maximal performance and eliminating
additional optimization steps
• Highly Reproducible Results: extensive testing and rigorous validation protocols minimize
lot-to-lot variation
• Assay Flexibility: custom conjugation is available for Alexa Fluor ® 488, 555, 594, or 647 dyes;
PE; Pacific Blue dye; Sepharose ® or magnetic beads; biotin; and HRP labels
Fluorescent Dye Antibody Conjugates
Fluorescent signal is produced when light energy from a laser or metal halide lamp at a specific
wavelength is absorbed by a fluorochrome (excitation) and then released (emission), producing light at
distinct wavelengths that can be measured by fluorescence detectors.
Fluorescent dyes can be used in IF to produce images of cell or tissue structures, and are often used
to study changes in protein localization and/or expression in response to a cell stimulus. Fluorescent
dye conjugates can be used in FC to analyze single cell signaling events, and are particularly useful
for multiplex assays because multiple primary antibodies conjugated to different fluorochromes can be
used to identify protein targets within a single sample regardless of host species, greatly increasing
assay flexibility.
Fluorescent dye conjugated antibody used
to visualize α-Synuclein expression
α-Synuclein (D37A6) XP ® Rabbit mAb (Alexa Fluor ® 488 Conjugate) #5496:
Confocal IF analysis of normal rat cerebellum using #5496 (green). Blue pseudocolor
= DRAQ5 ® #4084 (fluorescent DNA dye).
S/N Ratio
45
40
35
30
25
20
15
10
5
0
0 1 2 3 4 5
Concentration (µg/ml)
Multiplex analysis: decreased expression of Sox2 and
Oct-4 pluripotency markers by day 5 in RA-induced NTERA-2 cells
Sox2
A B C
Oct-4A
Fluorescent dye conjugated antibody used to detect
etoposide-induced apoptotic cell populations
Cleaved Caspase-3 (Asp175) (D3E9) Rabbit mAb (Pacific Blue Conjugate)
#8788: Flow cytometric analysis of Jurkat cells, untreated (blue) or etoposide-treated
(green), using #8788.
Events
Cleaved Caspase-3 (Asp175)
(Pacific Blue Conjugate)
Small Molecule, Enzyme, and Bead Conjugates
Conjugation of an antibody to the horseradish peroxidase (HRP) enzyme is a common strategy for
detecting proteins in cell lysates by WB or ELISA. HRP catalyzes the oxidation of luminescent or chromogenic
substrates into detectable light or colored products. Small molecule conjugates such as biotin
take advantage of the natural affinity between biotin and streptavidin and can be detected directly by
streptavidin-HRP. Conjugating an antibody to a Sepharose ® or magnetic bead allows for the physical
separation of the detected protein from a whole cell lysate by centrifugation or magnetic force. Bead
conjugates can be used to enrich low abundance proteins or to study protein-protein interactions using
an antibody to a target of interest and analyzing proteins that co-precipitate.
HRP enzyme labeled antibody
used for direct WB detection
kDa
80
60
50
40
30
20
1 2 3 4
Cytochrome c (D18C7) Rabbit
mAb (HRP Conjugate) #12959:
WB analysis of extracts from various
cell lines using #12959.
Lanes
1. HeLa 3. C2C12
2. NIH/3T3 4. C6
Oct-4A (C30A3) Rabbit mAb
(Alexa Fluor ® 488 Conjugate)
#5177: Flow cytometric analysis of
NTERA-2 cells, treated with 10 μM
retinoic acid (RA) for 5 days to induce
neuronal differentiation, using #5177
and Sox2 (D6D9) XP ® Rabbit mAb
(Alexa Fluor ® 647 Conjugate) #5067.
At 0 days (A), 3 days (B), and 5 days
(C) of treatment, cells were harvested,
fixed, and permeabilized according to
the CST standard flow protocol and
analyzed on a 4-laser Gallios Flow
Cytometer (Beckman Coulter).
10
Cytochrome c
Bead conjugates are used for protein enrichment by IP.
Fluorescent dye
conjugated antibody
used to detect
nuclear induction
of phospho-p53.
Phospho-p53 (Ser15) (16G8) Mouse
mAb (Alexa Fluor ® 647 Conjugate)
#8695: Confocal IF analysis of HT-29
cells, untreated (left) or UV-treated
(right), using #8695 (blue pseudocolor).
Actin filaments were labeled with
DyLight 554 Phalloidin #13054 (red).
Phospho-Stat3 (Tyr705) (D3A7) XP ® Rabbit mAb (Sepharose ® Bead Conjugate)
#4074: IP of HeLa cell lysates, untreated or treated with Human Interferon-α1 (hIFN-α1)
#8927, using XP ® Rabbit (DA1E) IgG (Sepharose ® Bead Conjugate) #3423 and #4074.
The WB was probed using Phospho-Stat3 (Tyr705) (3E2) Mouse mAb #9138.
Lanes
1. Rabbit (DA1E) mAb IgG XP ® Isotype Control
(Sepharose ® Bead Conjugate) #3423
2. Phospho-Stat3 (Tyr705) (D3A7) XP ® Rabbit mAb
(Sepharose ® Bead Conjugate) #4074
kDa
200
140
100
80
60
50
40
30
1
2
– + – +
Phospho-
Stat3
(Tyr705)
hIFN-α1
270 For Research Use Only. Not For Use in Diagnostic Procedures. See pages 302 & 303 for Pathway Diagrams, Application, and Reactivity keys.
www.cellsignal.com/localization
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