FAQ: 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.

Secondary Antibodies
ProductConjugateELISAWestern BlotHisto- / CytochemistryFlow Cytometry
Whole IgG and F(ab’)2Unconjugated10 - 20 µg/ml10 - 20 µg/ml10 - 20 µg/ml10 - 20 µg/ml
FabUnconjugated20 - 40 µg/ml20 - 40 µg/ml20 - 40 µg/ml20 - 40 µg/ml
Whole IgG, F(ab’)2 and FabAlexa Fluor 488, 594, 647 and Cy31:100 - 1:8001:100 - 1:800
Whole IgGAlexa Fluor 680 and 7901:50.000 - 1:200.000
Whole IgGBV421 and BV4801:50 - 1:2001:50 - 1:200
Whole IgG, F(ab’)2 and FabAMCA, Cy2, FITC, TRITC, RRX1:50 - 1:2001:50 - 1:200
Whole IgGCy51:100 - 1:4001:100 - 1:400
Whole IgG, F(ab’)2R-PE and APC1:50 - 1:200
Whole IgG, F(ab’)2PerCP1:25 - 1:100
Whole IgG and F(ab’)2Horseradish Peroxidase (HRP)1:5.000 - 1:100.0001:5.000 - 1:100.000 ; 1:10.000 - 1:200.000 (ECL)1:200 - 1:5.000
Whole IgG and F(ab’)2Alkaline Phosphatase1:5.000 - 1:50.0001:5.000 - 1:50.000
Whole IgG, F(ab’)2 and FabBiotin (using Fluorophore-conjugated Streptavidin)1:200 - 1:1.0001:200 - 1:1.000
Whole IgG and F(ab’)2Biotin (using Enzym-conjugated Streptavidin)1:20.000 - 1:40.0001:20.000 - 1:40.0001:500 - 1:5.0001:200 - 1:1.000
Streptavidin
ProductConjugateELISAWestern BlotHisto- / CytochemistryFlow Cytometry
StreptavidinR-PE and APC1:50 - 1:200
StreptavidinPerCP1:25 - 1:100
StreptavidinHorseradish Peroxidase (HRP)1 - 2 µg/ml1 - 2 µg/ml ; 0,01 - 1 µg/ml (ECL)1 - 2 µg/ml
StreptavidinAlkaline Phosphatase1 - 2 µg/ml1 - 2 µg/ml ; 0,1 - 1 µg/ml (ECL)1 - 2 µg/ml
StreptavidinAlle Alexa Fluor, DyLight 405 and Cy30,5 - 2 µg/ml0,5 - 2 µg/ml
StreptavidinCy51 - 4 µg/ml1 - 4 µg/ml
StreptavidinAlle other Fluorophores2 - 5 µg/ml2 - 5 µg/ml
Additional Products
ProductELISAWestern BlotHisto- / CytochemistryFlow Cytometry
4nm Colloidal Gold-Antikörper Komplex1:20 - 1:200
6nm, 12 Colloidal Gold-Antikörper Komplex1:20 - 1:40
18nm Colloidal Gold-Antikörper Komplex1:10 - 1:20
Peroxidase-Anti-Peroxidase (PAP)1:5.000 - 1:50.0001:5.000 - 1:50.00025 - 50 µg/ml, 1:400 - 1:800
ChromPure10 µg/ml
Blocking Reagent

To reduce non specific background signals we recommend to use 5% v/v of normal serum as blocking reagent.