Our second Flow lab FAQ is something we probably wish we were asked about more often...
Antibody Titration - How And Why?
Titration allows the identification of the optimal concentration of a given reagent in a particular experiment.
It's worth doing because sometimes, the concentration recommended by the company, or in a paper, is
not the best to use in your assay.
Why?
Titration will help you to find the best signal-to-noise ratio for your staining and therefore give you the best chance of detecting a weak signal. It may also save you money if the optimal concentration is less than the manufacturer's recommendation:
All fluorescence measurements have associated background noise, which comes mainly from two sources. Some intracellular molecules, like NADH, fluoresce when excited at certain wavelengths (autofluorescence). The flow cytometer also has inherent noise coming from detectors and electronics.
The background noise can increase if the concentration of the dye/antibody is too high, because it will start binding not only to the high-affinity epitopes it's supposed to target, but also to low affinity ones (non-specific binding). In practical terms, the median of the negative signal will increase causing a drop in the staining index. You can see this drop in the green arrow on the plot below.
How:
Stain a known number
of cells with a range of different antibody concentrations. For example, if the
recommended concentration is 10μM
you can test 2.5, 5, 10 and 20μM. Then analyse them on your flow cytometer. It is very important
that the staining conditions are kept the same in the titration and in the
experiment (incubation time, temperature, fixation, etc), because the staining
level is affected by experimental conditions. The volume of the reaction is also critical, try to keep this constant.
Which concentration
should I choose after the titration?
To
select which concentration (or range of concentrations) is the best to use,
calculate the STAIN INDEX for each tested value. The
stain index is calculated as follows:
The
concentration with highest stain index should be selected for use on the
experimental panel. In the graph above, the best range of concentrations
is marked with a red box.
Frequently Asked Questions
1.
Should
I use a viability dye in the titration?
If possible, yes. Dead cells usually have higher levels of autofluorescnce and can bind antibodies non-specifically.
2.
Can
I use compensation beads for the titration?
No! With titration we are looking for the specific binding of an antibody to antigen. Compensation beads work by binding the light chains and the antibody NOT the specific binding site.
3.
My
marker is really rare. Can I test the same fluorochrome targeting a more
abundant marker?
No! We are trying to establish the antibody binding NOT the level of fluorescence. If the marker is rare in your cells, try to find a cell line where it is highly expressed; manufacturers datasheets will often say which cells have been used to test the antibody.
Remember - It is important to keep the same staining conditions for the titration and for the final experiment.
Joana
No comments:
Post a Comment