I purchase primary PPARγ antibody (H-100) is a rabbit polyclonal IgG provided at 200 µg/ml and secondary goat anti-rabbit IgG-HRP: sc-2004 200 µg in 0.5 ml volume. How much I dilute to perform maximum number of test with these antibodies?
This depends on the antibody and you will have to titrate it. Typically we use primaries at 1:1000 to 1:2000 (but I have already used bad antibodies at 1:200), secondaries I have used from 1:5000 to 1:20.000. I would make a dilution series of your protein and then start with 1:1000 dilution (or whatever else the manufacturer recommends). If this gives a nice blot, go down with the concentration gradually to see how far you can get. BTW: You can re-use the primary, when you add sodium azide to the solution (not to the secondary, azide kills the conjugated enzyme).
This depends on the antibody and you will have to titrate it. Typically we use primaries at 1:1000 to 1:2000 (but I have already used bad antibodies at 1:200), secondaries I have used from 1:5000 to 1:20.000. I would make a dilution series of your protein and then start with 1:1000 dilution (or whatever else the manufacturer recommends). If this gives a nice blot, go down with the concentration gradually to see how far you can get. BTW: You can re-use the primary, when you add sodium azide to the solution (not to the secondary, azide kills the conjugated enzyme).
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