Are You Using Too Much Secondary Antibody?
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?
Table 1. Alexa Fluor® Dyes Require Only a Fraction of the Working Concentration of Other Dyes
|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|