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Should you titer antibodies?


We all use antibodies in western blotting, flow cytometry and IHC just to name a few applications. In each assay we use 1 uL or a 1:100 dilution, right? If that doesn’t work as we had anticipated, then we increase the amount. Hopefully that works. If it doesn’t work, then what do we do? Who has the time or extra samples to optimize the concentration?

So why titer?

Not all antibodies will work at 1 uL because:

  • Each antibody is its own unique protein,
  • With its own specific affinity for the particular antigen,
  • Which leads to each antibody having its own unique titration range.

Finding that titration “happy place” will make your analysis significantly easier and each experiment more robust.  Using too much can increase the background and reduce the ability to distinguish the positive population. This is demonstrated in the figure below where the antibody was used at 1 ug/sample.  As the antibody is titered, the background goes down and the positive staining is easily visualized. However, as you continue to titer lower (at 0.015 ug/sample), you begin to lose the positive signal. In this example, the happy place is at 0.06 ug/sample.  This is the concentration where the best signal to noise ratio is obtained.

                         1.0 ug                   0.25 ug                       0.06 ug           0.015 ug

Why titer your antibodies

eBioscience takes the guess work out of titration

  • Human antibodies: Most directly conjugated flow antibodies are provided as a test size, meaning they are prediluted to the optimal concentration for your use. Using the recommended 5 uL per sample (a sample is defined as a final staining volume of 100 uL) will provide the best signal to noise ratio for your staining.  For multicolor staining panels , simply add 5 uL of each conjugated antibody to your cells, incubate, wash and analyze.
  • Mouse antibodies: Because we know that you want flexibility, we provide our mouse antibodies in milligram concentrations. (They range from 0.2-0.5 mg/mL).  Our technical data sheet provides our recommendation for the optimal concentration of antibody to use in a staining protocol. This is the concentration that will provide the best signal to noise staining, and will provide an excellent starting place if you want to perform 2 or 4 fold serial dilutions of the antibody in order to optimize the signal to noise for your specific experiment/sample type.
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