Are You Using Too Much Secondary Antibody?
 | If you’re not using Alexa Fluor® dyes are you really getting the most for your money? To find out, we tested a number of secondary antibody conjugates to determine what working concentrations were needed to achieve optimal working conditions. |
Mitochondria of bovine pulmonary artery endothelial cells were labeled with an antibody against the complex V inhibitor protein of the oxidative phosphorylation system, followed by detection with the secondary antibody conjugate. All samples were processed equally and mounted in phosphate-buffered saline. Initial experiments were performed in triplicate to determine optimal concentration and incubation times.
These optimal conditions showed that many new secondary antibody conjugates required a much higher concentration to achieve performance comparable to the Alexa Fluor® 488 dye conjugate (Table 1).
So are you really getting premium performance for your research dollars—or simply using too much dye?
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Table 1. Alexa Fluor® Dyes Require Only a Fraction of the Working Concentration of Other Dyes
| Fluorophore* | Vendor | Working Concentration |
|---|---|---|
| Alexa Fluor® 488 | Invitrogen | 1 μg/mL |
| Chromeo™ 488 | Active Motif | 15 μg/mL |
| HiLyte Fluor™ 488 | AnaSpec | 25 μg/mL |
| MFP488 | Boca Scientific | 15 μg/mL |
| Northern Lights™ 493 | R&D Systems | 20 μg/mL |
| PromoFluor 488 | PromoKine | 15 μg/mL |
