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.
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.
My other passions include grilling, wine tasting, and real food. To be honest, my biggest passion is flow cytometry, which is something that Carol and I share. My personal mission is to make flow cytometry education accessible, relevant, and fun. I’ve had a long history in the field starting all the way back in graduate school.
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