Secondary antibodies from Jackson ImmunoResearch are used at the highest dilution for many immunoassays. Our recommendation for ELISA, Western Blot, Histo- / Cytochemistry and Flow Cytometry (FACS) are presented in the following table. Please note, the optimal dilution is dependent on several factors, each user should determine empirically the right working dilution for their experiment.
Secondary Antibodies
| Product | Conjugate | ELISA | Western Blot | Histo- / Cytochemistry | Flow Cytometry |
| Whole IgG and F(ab’)2 | Unconjugated | 10 – 20 µg/ml | 10 – 20 µg/ml | 10 – 20 µg/ml | 10 – 20 µg/ml |
| Fab | Unconjugated | 20 – 40 µg/ml | 20 – 40 µg/ml | 20 – 40 µg/ml | 20 – 40 µg/ml |
| Whole IgG, F(ab’)2 and Fab | Alexa Fluor 488, 594, 647 and Cy3 | 1:100 – 1:800 | 1:100 – 1:800 | ||
| Whole IgG | Alexa Fluor 680 and 790 | 1:50.000 – 1:200.000 | |||
| Whole IgG | BV421 and BV480 | 1:50 – 1:200 | 1:50 – 1:200 | ||
| Whole IgG, F(ab’)2 and Fab | AMCA, Cy2, FITC, TRITC, RRX | 1:50 – 1:200 | 1:50 – 1:200 | ||
| Whole IgG | Cy5 | 1:100 – 1:400 | 1:100 – 1:400 | ||
| Whole IgG, F(ab’)2 | R-PE and APC | 1:50 – 1:200 | |||
| Whole IgG, F(ab’)2 | PerCP | 1:25 – 1:100 | |||
| Whole IgG and F(ab’)2 | Horseradish Peroxidase (HRP) | 1:5.000 – 1:100.000 | 1:5.000 – 1:100.000 ; 1:10.000 – 1:200.000 (ECL) | 1:200 – 1:5.000 | |
| Whole IgG and F(ab’)2 | Alkaline Phosphatase | 1:5.000 – 1:50.000 | 1:5.000 – 1:50.000 | ||
| Whole IgG, F(ab’)2 and Fab | Biotin (using Fluorophore-conjugated Streptavidin) | 1:200 – 1:1.000 | 1:200 – 1:1.000 | ||
| Whole IgG and F(ab’)2 | Biotin (using Enzym-conjugated Streptavidin) | 1:20.000 – 1:40.000 | 1:20.000 – 1:40.000 | 1:500 – 1:5.000 | 1:200 – 1:1.000 |
Streptavidin
| Product | Conjugate | ELISA | Western Blot | Histo- / Cytochemistry | Flow Cytometry |
| Streptavidin | R-PE and APC | 1:50 – 1:200 | |||
| Streptavidin | PerCP | 1:25 – 1:100 | |||
| Streptavidin | Horseradish Peroxidase (HRP) | 1 – 2 µg/ml | 1 – 2 µg/ml ; 0,01 – 1 µg/ml (ECL) | 1 – 2 µg/ml | |
| Streptavidin | Alkaline Phosphatase | 1 – 2 µg/ml | 1 – 2 µg/ml ; 0,1 – 1 µg/ml (ECL) | 1 – 2 µg/ml | |
| Streptavidin | Alle Alexa Fluor, DyLight 405 and Cy3 | 0,5 – 2 µg/ml | 0,5 – 2 µg/ml | ||
| Streptavidin | Cy5 | 1 – 4 µg/ml | 1 – 4 µg/ml | ||
| Streptavidin | Alle other Fluorophores | 2 – 5 µg/ml | 2 – 5 µg/ml |
Additional Products
| Product | ELISA | Western Blot | Histo- / Cytochemistry | Flow Cytometry |
| 4nm Colloidal Gold-Antikörper Komplex | 1:20 – 1:200 | |||
| 6nm, 12 Colloidal Gold-Antikörper Komplex | 1:20 – 1:40 | |||
| 18nm Colloidal Gold-Antikörper Komplex | 1:10 – 1:20 | |||
| Peroxidase-Anti-Peroxidase (PAP) | 1:5.000 – 1:50.000 | 1:5.000 – 1:50.000 | 25 – 50 µg/ml, 1:400 – 1:800 | |
| ChromPure | 10 µg/ml |
Blocking Reagent
To reduce non specific background signals we recommend to use 5% v/v of normal serum as blocking reagent.