Jackson Immuno Research Inc.
specializing in secondary antibodies and conjugates

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Fluorescent Dyes

Brilliant Violet™
421 & 480

  • Combine with AF488, RR-X, & AF647 for 5-color imaging
  • Compatible with commonly used filter sets
  • Switch nuclear stain from DAPI to DRAQ5™ for additional labeling options

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Whole IgG Affinity-Purified Secondary Antibodies

"Without question, I can always turn to Jackson ImmunoResearch secondary antibodies for ECL detection of any primary antibody! They produce great signal at low dilutions."

W Thompson, OSHU

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Exhibit Schedule

9-10th Apr - SoCalFlow - S. California Flow Society, Beckman Center - UC Irvine, CA

12th Apr - Texas Medical Center, LSE - Houston, TX

15-18th Apr - AACR - Amer Assoc Cancer Research - Chicago, IL

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Sequential Labeling

Importance of Sequential Labeling
and Titrating Primary vs. Target Antigen

Figures 1-5 show how an optimized multiple labeling method can improve results. The potential for cross-reactive interactions of the FabuLights™ is minimized by sequentially labeling the less abundant target antigen first, and by titrating Fab-labeled complexes vs. target antigen.

Blood was collected from normal human donors and treated with lysis buffer. Cells were centrifuged, washed, and resuspended in isotonic PBS + 0.5% BSA.

Complexes were formed using Fab anti-Fc:primary at 3:1 ratio (equal weight ratios). Alexa Fluor® 488-conjugated Fab Goat Anti-Mouse IgG1, Fcγ fragment specific was complexed with Mouse Anti-Human CD3 (BD #555330)( AF488/CD3); and Alexa Fluor® 647-conjugated Fab Goat Anti-Mouse IgG1, Fcγ fragment specific was complexed with Mouse Anti-Human CD19 (BD #555410)(AF647/CD19).

Cells were incubated with complexed antibodies under various staining conditions for 30 minutes, washed, and analyzed on a BD FACSCalibur, gated on lymphocytes.


1. Cells incubated with AF488/CD3 and washed. A large population of T cells is visible in LR quadrant.

Left figure 1.0 μg anti-CD3/tube, right figure 0.5 μg anti-CD3/tube.


2. Cells incubated with AF647/CD19 and washed. A smaller population of B cells is visible in UL quadrant.

Left figure 1.0 μg anti-CD19/tube, right figure 0.1 μg anti-CD19/tube.


3. Cells incubated with a cocktail of AF488/CD3 and AF647/CD19 and washed.

Left figure: Anti-CD3 and anti-CD19 both at 1 μg of antibody/tube. Labeled Fab fragments are able to bind to incorrect primary antibody, resulting in unacceptable cross-talk. Most notably, excess AF647-FabuLight has bound to anti-CD3 on the T cells, shifting the T cell population into UR quadrant.
Right figure: Anti-CD3 is at 0.5 μg/tube, anti-CD19 is at 0.1 μg/tube. Titrated antibodies still result in cross-talk, though T cells have remained in the LR quadrant with insignificant loss of signal.


4. Cells incubated with AF647/CD19 (primary to less abundant target used first), washed, then incubated with AF488/CD3 and washed. Note specific labeling of T cells in LR quadrant and B cells in UL quadrant.

Left figure: Anti-CD3 and anti-CD19 both at 1 μg/tube. Right figure: Anti-CD3 is at 0.5 μg/tube, anti-CD19 is at 0.1 μg/tube. Titrated antibodies produce cleaner specific labeling with insignificant signal differences.


5. Cells incubated with AF488/CD3 (primary to more abundant target used first), washed, then incubated with AF647/CD19 and washed.

Left figure: Anti-CD3 and anti-CD19 both at 1 μg/tube. Excess AF647-FabuLight has bound to anti-CD3 on the T cells, shifting the population into UR quadrant.
Right figure: Anti-CD3 is at 0.5 μg/tube, anti-CD19 is at 0.1 μg/tube. T cells have shifted down. Further titration of anti-CD19 can improve this result, but labeling the less abundant primary first (as in #4) avoids the problem.