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Dilution of Secondary Antibodies

 

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.

 

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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

 

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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.