Using Blocking Buffer - LI-COR C-DiGit User Manual

Chapter 3: Scanning Membranes
Click here to watch a video that demonstrates how to place a membrane on
the C-DiGit Blot Scanner.

Using Blocking Buffer

1) Can I use Odyssey® blocking buffer to block my blot?
Yes. Use only for the blocking step and be aware that the sodium azide from the Odyssey block-
ing buffer may still be present on the membrane at the detection step and will bind to the HRP
enzyme, resulting in reduced light production and less intense bands.
2) Can I use milk-based blockers?
Yes. Milk-based blockers can be used for chemiluminescent detection but should be avoided
when detecting phosphoproteins or glycoproteins. Milk- based blockers may contain endo-
genous biotin and glycoproteins, resulting in higher background on the membrane.
3) Can I dilute the HRP-conjugated secondary antibodies in Odyssey® blocking buffer?
No. Odyssey blocking buffer contains sodium azide as a preservative. Sodium azide binds irre-
versibly to the HRP enzyme, inhibiting the binding of the substrate and slowing the
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