Editing Western blot (section)

== Applications ==
[[File:Western Blot results for HIV test.jpg|thumb|Western blot [[HIV test]] where the first two strips are negative and positive controls followed by actual tests]]
The western blot is extensively used in [[biochemistry]] for the qualitative detection of single proteins and protein-modifications (such as [[post-translational modification]]s). At least 8–9% of all protein-related publications are estimated to apply western blots.<ref name="Moritz2020" /> It is used as a general method to identify the presence of a specific single protein within a complex mixture of proteins. A semi-quantitative estimation of a protein can be derived from the size and colour intensity of a protein band on the blot membrane. In addition, applying a [[dilution series]] of a purified protein of known concentrations can be used to allow a more precise estimate of protein concentration. The western blot is routinely used for verification of [[protein production]] after [[cloning]]. It is also used in medical diagnostics, e.g., in the [[HIV test]] or [[Bovine spongiform encephalopathy|BSE]]-Test.<ref>{{cite journal | vauthors = Alexander TS | title = Human Immunodeficiency Virus Diagnostic Testing: 30 Years of Evolution | journal = Clinical and Vaccine Immunology | volume = 23 | issue = 4 | pages = 249–253 | date = April 2016 | pmid = 26936099 | pmc = 4820517 | doi = 10.1128/CVI.00053-16 | veditors = Pasetti MF }}</ref>

The confirmatory HIV test employs a western blot to detect anti-HIV antibody in a human [[blood plasma|serum]] sample. Proteins from known [[HIV]]-infected cells are separated and blotted on a membrane as above. Then, the serum to be tested is applied in the primary antibody incubation step; free antibody is washed away, and a secondary anti-human antibody linked to an enzyme signal is added. The stained bands then indicate the proteins to which the patient's serum contains antibody.<ref>{{cite journal | vauthors = Sudha T, Lakshmi V, Teja VD | title = Western blot profile in HIV infection | journal = Indian Journal of Dermatology, Venereology and Leprology | volume = 72 | issue = 5 | pages = 357–360 | date = 2006 | pmid = 17050930 | doi = 10.4103/0378-6323.27752 | doi-access = free }}</ref> A western blot is also used as the definitive test for [[variant Creutzfeldt–Jakob disease]], a type of prion disease linked to the consumption of contaminated beef from cattle with [[bovine spongiform encephalopathy]] (BSE, commonly referred to as 'mad cow disease').<ref>{{cite journal | vauthors = Ingrosso L, Vetrugno V, Cardone F, Pocchiari M | title = Molecular diagnostics of transmissible spongiform encephalopathies | journal = Trends in Molecular Medicine | volume = 8 | issue = 6 | pages = 273–280 | date = June 2002 | pmid = 12067613 | doi = 10.1016/S1471-4914(02)02358-4 }}</ref> Another application is in the diagnosis of [[tularemia]]. An evaluation of the western blot's ability to detect antibodies against ''[[F. tularensis]]'' revealed that its sensitivity is almost 100% and the specificity is 99.6%.<ref>{{cite journal | vauthors = Schmitt P, Splettstösser W, Porsch-Ozcürümez M, Finke EJ, Grunow R | title = A novel screening ELISA and a confirmatory Western blot useful for diagnosis and epidemiological studies of tularemia | journal = Epidemiology and Infection | volume = 133 | issue = 4 | pages = 759–766 | date = August 2005 | pmid = 16050523 | pmc = 2870305 | doi = 10.1017/s0950268805003742 | doi-broken-date = 1 November 2024 }}</ref> Some forms of [[Lyme disease]] testing employ western blotting.<ref>{{cite journal | vauthors = Artsob H | title = Western Blot as a confirmatory test for Lyme disease | journal = The Canadian Journal of Infectious Diseases | volume = 4 | issue = 2 | pages = 115–116 | date = March 1993 | pmid = 22346434 | pmc = 3250769 | doi = 10.1155/1993/796390 | doi-access = free }}</ref> A western blot can also be used as a confirmatory test for Hepatitis B infection and HSV-2 (Herpes Type 2) infection.<ref>{{cite journal | vauthors = De Castro L, Yoshida CF, Gaspar AM, Gomes SA | title = Western blot analysis of the reactivity between envelope proteins of hepatitis B viruses from Brazilian carriers and antibodies raised against recombinant hepatitis B vaccines | journal = Acta Virologica | volume = 40 | issue = 5–6 | pages = 251–258 | date = December 1996 | pmid = 9171452 | url = https://pubmed.ncbi.nlm.nih.gov/9171452/ | access-date = 6 December 2020 }}</ref><ref>{{cite journal | vauthors = Golden MR, Ashley-Morrow R, Swenson P, Hogrefe WR, Handsfield HH, Wald A | title = Herpes simplex virus type 2 (HSV-2) Western blot confirmatory testing among men testing positive for HSV-2 using the focus enzyme-linked immunosorbent assay in a sexually transmitted disease clinic | journal = Sexually Transmitted Diseases | volume = 32 | issue = 12 | pages = 771–777 | date = December 2005 | pmid = 16314775 | doi = 10.1097/01.olq.0000175377.88358.f3 | s2cid = 10591513 | doi-access = free }}</ref> In veterinary medicine, a western blot is sometimes used to confirm [[FIV]]+ status in cats.<ref>{{cite web |title=FIV testing – which to use |url=http://fivcats.com/FIV/fiv_testing.html |website=Catwork |access-date=6 December 2020}}</ref>

Further applications of the western blot technique include its use by the [[World Anti-Doping Agency]] (WADA). [[Blood doping]] is the misuse of certain techniques and/or substances to increase one's red blood cell mass, which allows the body to transport more oxygen to muscles and therefore increase stamina and performance.  There are three widely known substances or methods used for blood doping, namely, [[erythropoietin]] (EPO), [[synthetic oxygen carrier]]s and blood transfusions. Each is prohibited under WADA's List of Prohibited Substances and Methods. The western blot technique was used during the 2014 FIFA World Cup in the anti-doping campaign for that event.<ref name="BaumeJan2015">{{cite journal | vauthors = Baume N, Jan N, Emery C, Mandanis B, Schweizer C, Giraud S, Leuenberger N, Marclay F, Nicoli R, Perrenoud L, Robinson N, Dvorak J, Saugy M | display-authors = 6 | title = Antidoping programme and biological monitoring before and during the 2014 FIFA World Cup Brazil | journal = British Journal of Sports Medicine | volume = 49 | issue = 9 | pages = 614–622 | date = May 2015 | pmid = 25878079 | pmc = 4413745 | doi = 10.1136/bjsports-2015-094762 }}</ref>  In total, over 1000 samples were collected and analysed by Reichel, et al.<ref name="Reichel2016" /> in the WADA accredited [[Laboratory of Lausanne]], [[Switzerland]]. Recent research utilizing the western blot technique showed an improved detection of EPO in blood and urine based on novel Velum SAR precast horizontal gels optimized for routine analysis.<ref>{{Cite web| vauthors = Schwenke D |date=2015|title=Application Note: Improved detection of EPO in blood and urine based on novel Velum SAR precast horizontal gels optimized for routine analysis|url=https://precisionbiosystems.com/wp-content/uploads/2016/10/Application_Note_Improvements-for-EPO-detection_Schwenke_2015.pdf}}</ref> With the adoption of the horizontal SAR-PAGE in combination with the precast film-supported Velum SAR gels the discriminatory capacity of micro-dose application of rEPO was significantly enhanced.

=== Identification of protein localization across cells ===
For medication development, the identification of therapeutic targets, and biological research, it is essential to comprehend where proteins are located within a cell.<ref name=":3" /><ref>{{cite journal | vauthors = Lee YH, Tan HT, Chung MC | title = Subcellular fractionation methods and strategies for proteomics | journal = Proteomics | volume = 10 | issue = 22 | pages = 3935–3956 | date = November 2010 | pmid = 21080488 | doi = 10.1002/pmic.201000289 | s2cid = 29256675 }}</ref> The subcellular locations of proteins inside the cell and their functions are closely related. The relationship between protein function and localization suggests that when proteins move, their functions may change or acquire new characteristics. A protein's subcellular placement can be determined using a variety of methods. Numerous efficient and reliable computational tools and strategies have been created and used to identify protein subcellular localization.<ref>{{cite book | vauthors = Barberis E, Marengo E, Manfredi M | title = Proteomics Data Analysis | chapter = Protein Subcellular Localization Prediction | series = Methods in Molecular Biology | volume = 2361 | pages = 197–212 | date = 2021 | pmid = 34236663 | doi = 10.1007/978-1-0716-1641-3_12 | publisher = Springer US | isbn = 978-1-0716-1640-6 | s2cid = 235768807 | place = New York, NY | veditors = Cecconi D }}</ref> With the aid of subcellular fractionation methods, WB continues to be an important fundamental method for the investigation and comprehension of protein localization.<ref name=":3" />

=== Epitope mapping ===
Due to their various epitopes, antibodies have gained interest in both basic and clinical research. The foundation of antibody characterization and validation is epitope mapping. The procedure of identifying an antibody's binding sites (epitopes) on the target protein is referred to as "epitope mapping." Finding the binding epitope of an antibody is essential for the discovery and creation of novel vaccines, diagnostics, and therapeutics.<ref name=":3" /> As a result, various methods for mapping antibody epitopes have been created. At this point, western blotting's specificity is the main feature that sets it apart from other epitope mapping techniques. There are several application of western blot for epitope mapping on human skin samples, hemorrhagic disease virus.<ref name=":3" /><ref>{{cite journal | vauthors = Maier RH, Maier CJ, Rid R, Hintner H, Bauer JW, Onder K | title = Epitope mapping of antibodies using a cell array-based polypeptide library | journal = Journal of Biomolecular Screening | volume = 15 | issue = 4 | pages = 418–426 | date = April 2010 | pmid = 20233905 | doi = 10.1177/1087057110363821 | s2cid = 210137 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Kong D, Liu J, Jiang Q, Yu Z, Hu X, Guo D, Huang Q, Jiao M, Qu L | display-authors = 6 | title = Production, Characterization, and Epitope Mapping of Monoclonal Antibodies Against Different Subtypes of Rabbit Hemorrhagic Disease Virus (RHDV) | journal = Scientific Reports | volume = 6 | issue = 1 | pages = 20857 | date = February 2016 | pmid = 26878800 | pmc = 4754648 | doi = 10.1038/srep20857 | bibcode = 2016NatSR...620857K }}</ref>