Editing Western blot (section)

=== Detection and visualization ===
[[File:Western blot chemiluminescent detection.png|thumb|Western blot chemiluminescent detection|upright=1.5]]
After the unbound probes are washed away, the western blot is ready for detection of the probes that are labeled and bound to the protein of interest. In practical terms, not all westerns reveal protein only at one band in a membrane. Size approximations are taken by comparing the stained bands to that of the marker or ladder loaded during electrophoresis. The process is commonly repeated for a structural protein, such as [[actin]] or [[tubulin]], that should not change between samples. The amount of target protein is [[Normalization (statistics)|normalized]] to the structural protein to control between groups. A superior strategy is the normalization to the total protein visualized with [[trichloroethanol]]<ref>{{cite journal | vauthors = Stennert E, Arold R | title = [The double external auditory canal (author's transl)] | journal = Hno | volume = 21 | issue = 10 | pages = 293–296 | date = October 1973 | pmid = 4769330 }}</ref><ref>{{cite journal | vauthors = Gilda JE, Gomes AV | title = Stain-Free total protein staining is a superior loading control to β-actin for Western blots | journal = Analytical Biochemistry | volume = 440 | issue = 2 | pages = 186–188 | date = September 2013 | pmid = 23747530 | pmc = 3809032 | doi = 10.1016/j.ab.2013.05.027 }}</ref> or [[epicocconone]].<ref>{{cite journal | vauthors = Moritz CP, Marz SX, Reiss R, Schulenborg T, Friauf E | title = Epicocconone staining: a powerful loading control for Western blots | journal = Proteomics | volume = 14 | issue = 2–3 | pages = 162–168 | date = February 2014 | pmid = 24339236 | doi = 10.1002/pmic.201300089 | s2cid = 206368546 }}</ref> This practice ensures correction for the amount of total protein on the membrane in case of errors or incomplete transfers. (see [[western blot normalization]])

==== Colorimetric detection ====
The colorimetric detection method depends on incubation of the western blot with a substrate that reacts with the reporter enzyme (such as [[peroxidase]]) that is bound to the secondary antibody. This converts the soluble dye into an insoluble form of a different colour that precipitates next to the enzyme and thereby stains the membrane. Development of the blot is then stopped by washing away the soluble dye. Protein levels are evaluated through [[densitometry]] (how intense the stain is) or [[spectrophotometry]].

==== Chemiluminescent detection ====
Chemiluminescent detection methods depend on incubation of the western blot with a substrate that will luminesce when exposed to the reporter on the secondary antibody. The light is then detected by [[CCD camera]]s which capture a digital image of the western blot or photographic film. The use of film for western blot detection is slowly disappearing because of non linearity of the image (non accurate quantification). The image is analysed by densitometry, which evaluates the relative amount of protein staining and quantifies the results in terms of optical density. Newer software allows further data analysis such as molecular weight analysis if appropriate standards are used.

==== Radioactive detection ====

Radioactive labels do not require enzyme substrates, but rather, allow the placement of medical X-ray film directly against the western blot, which develops as it is exposed to the label and creates dark regions which correspond to the protein bands of interest (see image above). The importance of radioactive detections methods is declining due to its hazardous radiation {{Citation needed|date=August 2007}}, because it is very expensive, health and safety risks are high, and ECL (enhanced chemiluminescence) provides a useful alternative.

==== Fluorescent detection ====
[[File:Anti-lipoic acid immunoblot.png|thumb|Western blot using an anti-[[lipoic acid]] primary antibody and an [[infrared|IR]]-dye labelled secondary antibody in ''[[Leishmania]]'' major extracts]]
The [[Fluorescent tag|fluorescently labeled]] probe is excited by light and the emission of the excitation is then detected by a photosensor such as a CCD camera equipped with appropriate emission filters which captures a digital image of the western blot and allows further data analysis such as molecular weight analysis and a quantitative western blot analysis. Fluorescence is considered to be one of the best methods for quantification but is less sensitive than chemiluminescence.<ref>{{cite book | vauthors = Mathews ST, Plaisance EP, Kim T | chapter = Imaging Systems for Westerns: Chemiluminescence vs. Infrared Detection | title = Protein Blotting and Detection | series = Methods in Molecular Biology | volume = 536 | pages = 499–513 | year = 2009 | pmid = 19378087 | doi = 10.1007/978-1-59745-542-8_51 | publisher = Humana Press | isbn = 978-1-934115-73-2 }}</ref>