Editing Western blot (section)

=== Incubation ===
[[File:Western Blotting.png|thumb|upright=1.5|Probing process]]
During the detection process, the membrane is "probed" for the protein of interest with a modified antibody which is linked to a reporter enzyme; when exposed to an appropriate substrate, this enzyme drives a colorimetric reaction and produces a colour. For a variety of reasons, this traditionally takes place in a two-step process, although there are now one-step detection methods available for certain applications.

==== Primary antibody ====
The [[Primary antibody|primary antibodies]] are generated when a host species or immune cell culture is exposed to the protein of interest (or a part thereof). Normally, this is part of the immune response, whereas here they are harvested and used as sensitive and specific detection tools that bind the protein directly.

After blocking, a solution of primary antibody (generally between 0.5 and 5 micrograms/mL) diluted in either PBS or TBST wash buffer is incubated with the membrane under gentle agitation for typically an hour at room temperature, or overnight at 4'''°'''C. It can also be incubated at different temperatures, with lesser temperatures being associated with more binding, both specific (to the target protein, the "signal") and non-specific ("noise"). Following incubation, the membrane is washed several times in wash buffer to remove unbound primary antibody, and thereby minimize background.<ref name=":0" /> Typically, the wash buffer solution is composed of buffered saline solution with a small percentage of detergent, and sometimes with powdered milk or BSA.

==== Secondary antibody ====
After rinsing the membrane to remove unbound primary antibody, the membrane is exposed to another antibody known as the [[Primary and secondary antibodies|secondary antibody]]. Antibodies come from animal sources (or animal sourced [[hybridoma]] cultures). The secondary antibody recognises and binds to the species-specific portion of the primary antibody.  Therefore, an anti-mouse secondary antibody will bind to almost any mouse-sourced primary antibody, and can be referred to as an 'anti-species' antibody (e.g. anti-mouse, anti-goat etc.). To allow detection of the target protein, the secondary antibody is commonly linked to [[biotin]] or a reporter [[enzyme]] such as [[alkaline phosphatase]] or [[horseradish peroxidase]]. This means that several secondary antibodies will bind to one primary antibody and enhance the signal, allowing the detection of proteins of a much lower concentration than would be visible by SDS-PAGE alone.

Horseradish peroxidase is commonly linked to secondary antibodies to allow the detection of the target protein by [[chemiluminescence]]. The chemiluminescent substrate is cleaved by horseradish peroxidase, resulting in the production of [[luminescence]]. Therefore, the production of luminescence is proportional to the amount of horseradish peroxidase-conjugated secondary antibody, and therefore, indirectly measures the presence of the target protein. A sensitive sheet of photographic film is placed against the membrane, and exposure to the light from the reaction creates an image of the antibodies bound to the blot. A cheaper but less sensitive approach utilizes a [[4-chloronaphthol]] stain with 1% [[hydrogen peroxide]]; the reaction of peroxide radicals with 4-chloronaphthol produces a dark purple stain that can be photographed without using specialized photographic film.

As with the [[ELISPOT]] and [[ELISA]] procedures, the enzyme can be provided with a substrate molecule that will be converted by the enzyme to a coloured reaction product that will be visible on the membrane (see the figure below with blue bands).

Another method of secondary antibody detection utilizes a near-infrared fluorophore-linked antibody. The light produced from the excitation of a fluorescent dye is static, making fluorescent detection a more precise and accurate measure of the difference in the signal produced by labeled antibodies bound to proteins on a western blot.  Proteins can be accurately quantified because the signal generated by the different amounts of proteins on the membranes is measured in a static state, as compared to chemiluminescence, in which light is measured in a dynamic state.<ref name=Ambroz2006 />

A third alternative is to use a radioactive label rather than an enzyme coupled to the secondary antibody, such as labeling an antibody-binding protein like ''[[Staphylococcus]]'' Protein A or Streptavidin with a radioactive isotope of iodine. Since other methods are safer, quicker, and cheaper, this method is now rarely used; however, an advantage of this approach is the sensitivity of auto-radiography-based imaging, which enables highly accurate protein quantification when combined with optical software (e.g. Optiquant).

==== One step ====
Historically, the probing process was performed in two steps because of the relative ease of producing primary and secondary antibodies in separate processes. This gives researchers and corporations huge advantages in terms of flexibility, reduction of cost, and adds an amplification step to the detection process. Given the advent of high-throughput protein analysis and lower limits of detection, however, there has been interest in developing one-step probing systems that would allow the process to occur faster and with fewer consumables. This requires a probe antibody which both recognizes the protein of interest and contains a detectable label, probes which are often available for known [[protein tags]]. The primary probe is incubated with the membrane in a manner similar to that for the primary antibody in a two-step process, and then is ready for direct detection after a series of wash steps.