Protein Detection using Antibody

Detection of the protein of interest is made possible by using specific antibodies. Before antibodies are applied to the membrane, a blocking solution, typically 5% bovine serum albumin (BSA) or de-fatted dried milk, is added to prevent nonspecific protein binding.

Generally, two antibodies are used in Western blots: primary antibodies and secondary antibodies. The primary antibodies have a unique binding site that can recognize specific proteins. After the membrane is incubated with primary antibodies and unbound primary antibodies are washed away, the secondary antibodies are added. The secondary antibodies will bind selectively to the primary antibodies. The secondary antibodies serve not only as a carrier of the detection label but also as an amplifier of the emitted signal. The label attached to the secondary antibodies is usually a biotin or a reporter enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP).

At the bottom of the image is a yellow rectangular membrane. To the membrane, a green molecule is attached, called target antibody. To the antibody, a red Y-shaped molecule is attached with arms pointing towards the target protein, called primary antibody. To the leg part of the red Y-shaped molecule, another blue Y-shaped molecule is attached, in similar fashion. To the leg part of the blue Y-shaped molecule, a red sphere is attached. The sphere together with the blue molecule are called enzyme-conjugated secondary antibody. Two arrows point towards the red sphere, one comes from small yellow spheres called detection signal and the other from light blue small spheres called enzyme substrate.

Figure 1. Protein detection using antibody.

Wide varieties of secondary antibodies with different labeling systems are available in four detection systems: colorimetric, chemiluminescence, radioactive and fluorescent.

As with all experiments, it is important to do add an experimental control. In Western blot, this is to test whether a similar amount of sample was added and thus to ensure whether the results can be compared. The most common control is actin. This protein is expressed at high levels in all cells and can be reliably visualized on a Western blot.

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