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everyone. I've just been introduced to the procedure of Western blotting from my reading, though I'm not entirely sure about some points. I'd appreciate it if someone could help me with this.

What exactly is the function of the blocking buffer?

Why does one need a secondary antibody?

When discussing nitrocellulose membranes, say of a 0.45 5m pore size, what does 5m refer to?

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Blocking buffer

Once the proteins in the gel have been transferred to the nitrocellulose membrane it is necessary to coat the rest of the surface of the membrane with an unrelated protein. This is necessary because all proteins will bind non-specifically to the nitrocellulose. Once the membrane has been blocked the only way that antibiody proteins (the probe) will bind to the membrane is via the transferred proteins that they recognise. The usual source of a blocking protein is defatted milk powder, i.e. casein, although some people use (or used to use) bovine serum albumin.

Secondary antibody

In a typical blotting experiment you expose first with a primary antibody directed against the protein of interest, for example this might be an IgG raised in a rabbit. Then in a second step you add a secondary antibody which will bind to the first one - in this case this might be goat ant-rabbit IgG (i.e. anti-rabbit IgG raised in a goat). Why is this done? The secondary antibody is in the form of an antibody-enzyme conjugate, typically with horseradish peoxidase, and the final development of the blot detects the presence of the conjugated enzyme.

Why use a secondary antibody? Two reasons: - firstly, there will sometimes be a degree of amplification if more than one secondary antibody molecule binds each primary antibody molecule; but more important is that it means that it is not necessary to produce an enzyme conjugated form of every primary antibody, which is time-consuming and quite tricky. So in your work you may be studying several different proteins using Westerns, but you will be able to use the same secondary antibody to detect all of them.

Pore size

That should be 0.45 - 0.5 µm. Pore sizes are measured in micrometres.

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