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Secondary Antibodies

For VHH Discovery

Anti-Alpaca IgG, Subclass and VHH domain secondaries

Ideally time PBMC harvest

Enhance detection of VHH antibodies

Test for VHH expression and binding activity

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Whole IgG Affinity-Purified Secondary Antibodies

"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

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Suggested Dilution Factors

Technical Service e-mail
tech@jacksonimmuno.com

TechFAQ #5

What are the suggested dilution factors to be used for Jackson ImmunoResearch products?

The dilution factors suggested in the following table are presented in the form of a range because the optimal dilution is a function of many factors, such as antigen density, permeability, etc. The actual dilution used must be determined empirically. Dilutions outside these ranges may be required occasionally, and should be determined based on actual circumstances.

Product	ELISA	Western Blots	Histo-/ Cyto-Chemistry	Flow Cytometry
Unconjugated whole IgG and F(ab') ₂ secondary antibodies	10-20 µg / ml
Unconjugated Fab secondary antibodies	20-40 µg / ml
Most Alexa Fluor®, DyLight, and Cy3 Conjugates [IgG, F(ab') ₂, Fab]			1:100- 1:800	1:100- 1:800
Alexa Fluor® 680 and 790 Conjugates		1:50,000-200,000
AMCA, Cy2, FITC, TRITC, RRX, and Texas Red Conjugates [IgG, F(ab') ₂, Fab]			1:50- 1:200	1:50- 1:200
Cy5 Conjugates [IgG, F(ab') ₂, Fab]			1:100- 1:400	1:100- 1:400
Phycoerythrin and Allophycocyanin Conjugates [IgG, F(ab') ₂, Streptavidin]				1:50- 1:200
PerCP Conjugates [IgG, F(ab') ₂, Streptavidin]				1:25- 1:100
Horseradish Peroxidase Conjugates [IgG, F(ab') ₂]	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
Alkaline Phosphatase Conjugates [IgG, F(ab') ₂]	1:5,000- 1:50,000	1:5,000- 1:50,000
Biotin-SP Conjugates [IgG, F(ab') ₂] (using Fluorophore-Conjugated Streptavidin)			1:200- 1:1,000	1:200 1:1,000
Biotin-Sp Conjugates [IgG, F(ab') ₂] (using enzyme-Conjugated Streptavidin)	1:20,000- 1:400,000	1:20,000- 1:400,000	1:500- 1:5,000
Horseradish Peroxidase-Conjugated Streptavidin	1-2 µg / ml	0.01-0.1 µg / ml	1-2 µg / ml
Alkaline Phosphatase-Conjugated Streptavidin	1-2 µg / ml	0.1-1µg / ml	1-2 µg / ml
All Alexa Fluor®-, DyLight 405-, and Cy3-Conjugated Streptavidin			0.5-2 µg / ml	0.5-2 µg / ml
Cy5-Conjugated Streptavidin			0.5-2 µg / ml	0.5-2 µg / ml
All Other Fluorophore-Conjugated Streptavidin			2-5 µg / ml	2-5 µg / ml
4 nm Gold-Antibody Complexes			1:20-1:200
18 nm Gold-Antibody Complexes			1:10-1:20
6, 12 nm Gold-Antibody Complexes			1:20-1:40
Peroxidase-Anti-Peroxidase (PAP)			25-50 µg / ml