"I have used a wide variety of secondaries and Jackson ImmunoResearch has consistently been the best. The fluorophores are bright and stable and their selective (x reactivity removed) secondaries have always shown species specificity in multiple labeling."
Janet Duerr, Ohio University
|
Technical Service e-mail
tech@jacksonimmuno.com |
The dilution factors suggested in the following table are presented as ranges because the optimal dilution is a function of many factors, such as antigen density, permeability, etc. The optimal working dilution should be determined empirically for each application.
| Application | |||||
|---|---|---|---|---|---|
| Product | Conjugate | ELISA |
Western
Blots |
Histo-/Cyto
chemistry |
Flow
Cytometry |
| Whole IgG and F(ab')2 secondary antibodies | Unconjugated | 10-20 µg/ml | |||
| Fab secondary antibodies | Unconjugated | 20-40 µg/ml | |||
| Whole IgG, F(ab')2 and Fab fragment secondary antibodies | Alexa Fluor® 488, 594, 647, DyLight™ 405, and Cy™3 | 1:100-1:800 | |||
| Whole IgG secondary antibodies | Alexa Fluor® 680 and 790 | 1:50,000-1:200,000 | |||
| Whole IgG, F(ab')2 and Fab fragment secondary antibodies | AMCA, BV421™, BV480™, Cy2, FITC, TRITC, RRX, and Texas Red | 1:50-1:200 | |||
| Whole IgG secondary antibodies | Cy™5 | 1:100-1:400 | |||
| Whole IgG, F(ab')2 secondary antibodies and Streptavidin | R-PE and APC | 1:50-1:200 | |||
| Whole IgG, F(ab')2 secondary antibodies and Streptavidin | PerCP | 1:25-1:100 | |||
| Whole IgG and F(ab')2 secondary antibodies | Horseradish Peroxidase | 1:5,000-1:100,000 |
1:5,000-1:100,000
(non-ECL) 1:10,000-1:200,000 (ECL) |
1:500-
1:5,000 |
|
| Whole IgG and F(ab')2 secondary antibodies | Alkaline Phosphatase | 1:5,000-1:50,000 | 1:5,000-1:50,000 | ||
| Whole IgG, F(ab')2 and Fab secondary antibodies | Biotin-SP (using Fluorophore-Conjugated Streptavidin) | 1:200-1:1,000 | 1:200-1:1,000 | ||
| Whole IgG and F(ab')2 secondary antibodies | Biotin-SP (using enzyme-Conjugated Streptavidin) | 1:20,000-1:400,000 | 1:20,000-1:400,000 | 1:500-1:5,000 | |
| Streptavidin | Horseradish Peroxidase | 1-2 µg/ml | 0.01-0.1 µg/ml | 1-2 µg/ml | |
| Streptavidin | Alkaline Phosphatase | 1-2 µg/ml | 0.1-1 µg/ml | 1-2 µg/ml | |
| Streptavidin | Alexa Fluors®, DyLight 405, and Cy3 | 0.5-2 µg/ml | 0.5-2 µg/ml | ||
| Streptavidin | Cy5 | 0.5-2 µg/ml | 0.5-2 µg/ml | ||
| Streptavidin | All Other Fluorophores | 2-5 µg/ml | 2-5 µg/ml | ||
| 4 nm Gold-Antibody Complexes | 1:20-1:200 | ||||
| 6, 12 nm Gold-Antibody Complexes | 1:20-1:40 | ||||
| 18 nm Gold-Antibody Complexes | 1:10-1:20 | ||||
| Peroxidase-Anti-Peroxidase (PAP) | 25-50 µg/ml | ||||
| FabuLight™ Anti-IgM | To complex with primary antibody in solution, use 1:1 weight ratio of Fab: primary antibody (15:1 molar ratio). | ||||
| FabuLight Anti-IgG | To complex with primary antibody in solution, use 1:1 weight ratio of Fab: primary antibody (3:1 molar ratio). | ||||
| Normal Serum | 5% (v/v) for blocking | ||||
| ChromPure™ | 10 µg/ml | ||||
