What Is Titration

Titration is the process of identifying the best concentration to use an antibody for a given assay. While the vendor will provide a specific concentration to use, this may not be appropriate for your assay.

Performing titration is a simple process: fix the cell concentration, the time of incubation, the volume of reaction and temperature. The below data will help you understand what is titration. The graph displays an antibody from Leinco Technologies () that was used to stain 1×106 cells for 20 minutes on ice.

To identify the best concentration to use, the modified Staining Index was calculated (see Telford et al., (2009) Cytometry A 75A:1031) and plotted against the concentration, as shown below.

Titration

As is shown by this figure, as the concentration increases above 0.5 μg/ml, the SI decreases, due in part to the increase in the background (non-specific staining).  At concentrations below 0.25  μg/ml, the SI decreases because the antibody is no longer at a saturating concentration.  Thus the best concentration to use is between 0.25-0.5 μg/ml.

Titration helps save money and reagents, ensures the optimal concentration of reagent is being used, and avoids background due to high concentration of Abs.

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Tim Bushnell, PhD
Tim Bushnell, PhD

Tim Bushnell holds a PhD in Biology from the Rensselaer Polytechnic Institute. He is a co-founder of—and didactic mind behind—ExCyte, the world’s leading flow cytometry training company, which organization boasts a veritable library of in-the-lab resources on sequencing, microscopy, and related topics in the life sciences.

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