Okay, it’s football season, and I thought the analogy fit. 🙂 Seriously, the right Blocking Buffer is critical to getting that great chemiluminescent Western blot.
Incubating the membrane in blocking buffer after the transfer step will result in enhanced sensitivity of your blot. Blocking buffer contains proteins that stick to the membrane, promoting specific binding of the primary antibody and minimizing non-specific interactions. Various blocking buffers are available, and it’s important to try several blockers to find the optimal solution for each antigen and antibody pair. There is not a best blocker for all conditions – so you will need to do some testing.
One very, very, very important thing to keep in mind is that the blocker used with HRP-conjugated secondary antibodies in the secondary antibody incubation step of chemiluminescent Western blotting cannot contain sodium azide.
Why?, you ask.
Well, sodium azide binds irreversibly to the HRP enzyme, inhibiting the binding of the substrate and slowing the chemiluminescent reaction. This results in less light production that may affect the appearance of less intense bands or even the entire blot. See the figures below – the blot on the left was done with blocker that did not contain azide; the Western blot on the right used Western blocking buffer with azide.
Nota bene: Odyssey® Blocking Buffer (PBS or TBS) (which does contain sodium azide) CAN be used to block the blot and to dilute the primary antibody but not to block or dilute in the secondary antibody incubation step when using HRP-conjugated secondary antibodies.
On Another Note: Milk is a common blocking buffer; however, milk-based blockers that contain endogenous biotin and glycoproteins may result in higher background on the membrane when detecting with streptavidin. Milk may also contain active phosphatases that can de-phosphorylate phosphoproteins on the membrane.
Go Digital with the Odyssey Fc Chemiluminescent and Infrared Fluorescent Imaging System or the C-DiGit® Chemiluminescent Western Blot Scanner!