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.
Suggested Dilution Factors
| 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, 555, 568, 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, Fab and AffiniPure-VHH™ Secondary Antibodies | Biotin-SP (using Fluorophore-Conjugated Streptavidin) | 1:200 – 1:1,000 | 1:200 – 1:1,000 | ||
| Whole IgG, F(ab’)2 and AffiniPure-VHH™ 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 | |
| AffiniPure-VHH™ Secondary Antibodies | Cy3, DyLight 405, Alexa Fluors® | 1:200 – 1:800 | |||
| AffiniPure-VHH™ Secondary Antibodies | Rhodamine Red-X | 1:100 – 1:400 | |||
| 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 | ||
| Whole IgG secondary antibodies | 4 nm Gold-Antibody Complexes | 1:20 – 1:200 | |||
| Whole IgG secondary antibodies | 6, 12 nm Gold-Antibody Complexes | 1:20 – 1:40 | |||
| Whole IgG secondary antibodies | 18 nm Gold-Antibody Complexes | 1:10 – 1:20 | |||
| Whole IgG secondary antibodies, Streptavidin and ChromPure | 40 nm Gold-Antibody Complexes | 1:10 – 1:100 | |||
| 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 | ||||
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