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The protein composition of snake venoms has been frequently analyzed by means of electrophoretic techniques, but the interaction of proteins in venoms has barely been addressed. An electrophoretic technique that has gained prominence to study this type of interaction is blue native polyacrylamide gel electrophoresis (BN-PAGE), which allows for the high-resolution separation of proteins in their native form. These protein complexes can be further discriminated by a second-dimension gel electrophoresis (SDS-PAGE) from lanes cut from BN-PAGE. Once there is no study on the use of bidimensional BN\/SDS-PAGE with snake venoms, this study initially standardized the BN\/SDS-PAGE technique in order to evaluate protein interactions in Bothrops atrox, Bothrops erythromelas, and Bothrops jararaca snake venoms. Results of BN\/SDS-PAGE showed that native protein complexes were present, and that snake venom metalloproteinases and venom serine proteinases maintained their enzymatic activity after BN\/SDS-PAGE. C-type lectin-like proteins were identified by Western blotting. Therefore, bidimensional BN\/SDS-PAGE proved to be an easy, practical, and efficient method for separating functional venom proteins according to their assemblage in complexes, as well as to analyze their biological activities in further details.<\/jats:p>","DOI":"10.3390\/toxins14100661","type":"journal-article","created":{"date-parts":[[2022,9,29]],"date-time":"2022-09-29T01:23:16Z","timestamp":1664414596000},"page":"661","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Two-Dimensional Blue Native\/SDS Polyacrylamide Gel Electrophoresis for Analysis of Brazilian Bothrops Snake Venoms"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3525-0224","authenticated-orcid":false,"given":"Natacha","family":"Ferreira de Oliveira","sequence":"first","affiliation":[{"name":"Laborat\u00f3rio de Fisiopatologia, Instituto Butantan, S\u00e3o Paulo 05503-900, SP, Brazil"},{"name":"Escola Superior do Instituto Butantan (ESIB), Instituto Butantan, S\u00e3o Paulo 05503-900, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ana Teresa Azevedo","family":"Sachetto","sequence":"additional","affiliation":[{"name":"Laborat\u00f3rio de Fisiopatologia, Instituto Butantan, S\u00e3o Paulo 05503-900, SP, Brazil"},{"name":"Escola Superior do Instituto Butantan (ESIB), Instituto Butantan, S\u00e3o Paulo 05503-900, SP, Brazil"},{"name":"Programa de P\u00f3s-Gradua\u00e7\u00e3o em Ci\u00eancias M\u00e9dicas, Faculdade de Medicina, Universidade de S\u00e3o Paulo, S\u00e3o Paulo 01246-000, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6364-3757","authenticated-orcid":false,"given":"Marcelo Larami","family":"Santoro","sequence":"additional","affiliation":[{"name":"Laborat\u00f3rio de Fisiopatologia, Instituto Butantan, S\u00e3o Paulo 05503-900, SP, Brazil"},{"name":"Escola Superior do Instituto Butantan (ESIB), Instituto Butantan, S\u00e3o Paulo 05503-900, SP, Brazil"},{"name":"Programa de P\u00f3s-Gradua\u00e7\u00e3o em Ci\u00eancias M\u00e9dicas, Faculdade de Medicina, Universidade de S\u00e3o Paulo, S\u00e3o Paulo 01246-000, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,9,23]]},"reference":[{"key":"ref_1","unstructured":"(2022, January 25). Sistema de Informa\u00e7\u00e3o de Agravos de Notifica\u00e7\u00e3o (Sinan), Available online: www.saude.gov.br\/sinanweb."},{"key":"ref_2","unstructured":"Campbell, J.A., and Lamar, W.W. (2004). The Venomous Reptiles of the Western Hemisphere, Comstock Publishing Associates."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1016\/S0742-8413(98)10079-8","article-title":"Comparison of the biological activities in venoms from three subspecies of the south american rattlesnake (crotalus durissus terrificus, c. Durissus cascavella and c. Durissus collilineatus)","volume":"122","author":"Santoro","year":"1999","journal-title":"Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1443","DOI":"10.1016\/j.toxicon.2010.08.011","article-title":"Comparative analysis of newborn and adult bothrops jararaca snake venoms","volume":"56","author":"Antunes","year":"2010","journal-title":"Toxicon"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"345","DOI":"10.1007\/s00204-020-02896-y","article-title":"A lipidomics approach reveals new insights into crotalus durissus terrificus and bothrops moojeni snake venoms","volume":"95","author":"Acunha","year":"2021","journal-title":"Arch. Toxicol."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Villar-Briones, A., and Aird, S.D. (2018). Organic and peptidyl constituents of snake venoms: The picture is vastly more complex than we imagined. Toxins, 10.","DOI":"10.3390\/toxins10100392"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Eble, J.A. (2019). Structurally robust and functionally highly versatile-c-type lectin (-related) proteins in snake venoms. Toxins, 11.","DOI":"10.3390\/toxins11030136"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1016\/j.crtox.2021.02.006","article-title":"Using yeast two-hybrid system and molecular dynamics simulation to detect venom protein-protein interactions","volume":"2","author":"Jia","year":"2021","journal-title":"Curr. Res. Toxicol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1016\/j.bbapap.2004.07.007","article-title":"Factor x activator from vipera lebetina venom is synthesized from different genes","volume":"1702","author":"Siigur","year":"2004","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1007\/BF00517389","article-title":"Neurotoxic and myotoxic effects of crotalus phospholipase a and its complex with crotapotin","volume":"292","author":"Breithaupt","year":"1976","journal-title":"Naunyn Schmiedebergs Arch. Pharmacol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Tasoulis, T., and Isbister, G.K. (2017). A review and database of snake venom proteomes. Toxins, 9.","DOI":"10.3390\/toxins9090290"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"e20200018","DOI":"10.1590\/1678-9199-jvatitd-2020-0018","article-title":"Venom complexity of bothrops atrox (common lancehead) siblings","volume":"26","author":"Hatakeyama","year":"2020","journal-title":"J. Venom. Anim. Toxins Incl. Trop. Dis."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1016\/j.jprot.2009.07.013","article-title":"Snake venomics and antivenomics of bothrops atrox venoms from colombia and the amazon regions of brazil, peru and ecuador suggest the occurrence of geographic variation of venom phenotype by a trend towards paedomorphism","volume":"73","author":"Cid","year":"2009","journal-title":"J. Proteom."},{"key":"ref_14","first-page":"73","article-title":"Combined venomics, venom gland transcriptomics, bioactivities, and antivenomics of two bothrops jararaca populations from geographic isolated regions within the brazilian atlantic rainforest","volume":"135","author":"Pla","year":"2015","journal-title":"J. Proteom."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"214","DOI":"10.1016\/j.jprot.2016.06.029","article-title":"An in-depth snake venom proteopeptidome characterization: Benchmarking bothrops jararaca","volume":"151","author":"Nicolau","year":"2017","journal-title":"J. Proteom."},{"key":"ref_16","first-page":"93","article-title":"Venomics and antivenomics of bothrops erythromelas from five geographic populations within the caatinga ecoregion of northeastern brazil","volume":"114C","author":"Jorge","year":"2014","journal-title":"J. Proteom."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Mackessy, S.P. (2021). Snake venom metalloproteinases. Handbook of Venoms and Toxins of Reptiles, Taylor & Francis. [2nd ed.].","DOI":"10.1201\/9780429054204"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"969","DOI":"10.1016\/j.toxicon.2005.02.012","article-title":"Structural considerations of the snake venom metalloproteinases, key members of the m12 reprolysin family of metalloproteinases","volume":"45","author":"Fox","year":"2005","journal-title":"Toxicon"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1016\/S0147-9563(96)80046-X","article-title":"Ancrod: The use of snake venom in the treatment of patients with heparin-induced thrombocytopenia and thrombosis undergoing coronary artery bypass grafting: Nursing management","volume":"25","author":"Lathan","year":"1996","journal-title":"Heart Lung"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"716778","DOI":"10.3389\/fneur.2021.716778","article-title":"Use of batroxobin in central and peripheral ischemic vascular diseases: A systematic review","volume":"12","author":"Lan","year":"2021","journal-title":"Front. Neurol."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Mackessy, S.P. (2021). Snake venom phospholipase a2 toxins. Handbook of Venoms and Toxins of Reptiles, Taylor & Francis. [2nd ed.].","DOI":"10.1201\/9780429054204"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"510","DOI":"10.1016\/j.jprot.2011.01.003","article-title":"Snake population venomics and antivenomics of bothrops atrox: Paedomorphism along its transamazonian dispersal and implications of geographic venom variability on snakebite management","volume":"74","author":"Calvete","year":"2011","journal-title":"J. Proteom."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"512","DOI":"10.1016\/j.toxicon.2012.03.001","article-title":"C-type lectin-like proteins from snake venoms","volume":"60","author":"Arlinghaus","year":"2012","journal-title":"Toxicon"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"14109","DOI":"10.1016\/S0021-9258(19)49685-3","article-title":"Coagulation factor x activating enzyme from russell\u2019s viper venom (rvv-x): A novel metalloproteinase with disintegrin (platelet aggregation inhibitor)-like and c-type lectin-like domains","volume":"267","author":"Takeya","year":"1992","journal-title":"J. Biol. Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"991","DOI":"10.1093\/oxfordjournals.jbchem.a021862","article-title":"Purification and characterization of a Ca2+ -dependent prothrombin activator, multactivase, from the venom of echis multisquamatus","volume":"122","author":"Yamada","year":"1997","journal-title":"J. Biochem."},{"key":"ref_26","first-page":"18","article-title":"Purification and characterization of ecamulin\u2014A prothrombin activator from the venom of multi-scaled viper (echis multisquamatus)","volume":"68","author":"Solovjov","year":"1996","journal-title":"Ukr Biokhim Zh 1978"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"309","DOI":"10.1016\/S0049-3848(97)00258-2","article-title":"Purification and characterization of multisquamase, the prothrombin activator present in echis multisquamatus venom","volume":"88","author":"Petrovan","year":"1997","journal-title":"Thromb. Res."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"5200","DOI":"10.1074\/jbc.271.9.5200","article-title":"Isolation and characterization of carinactivase, a novel prothrombin activator in echis carinatus venom with a unique catalytic mechanism","volume":"271","author":"Yamada","year":"1996","journal-title":"J. Biol. Chem."},{"key":"ref_29","first-page":"796128","article-title":"Complexome profiling-exploring mitochondrial protein complexes in health and disease","volume":"9","author":"Potter","year":"2021","journal-title":"Front. Cell Dev. Biol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"103990","DOI":"10.1016\/j.jprot.2020.103990","article-title":"From classical to new generation approaches: An excursus of -omics methods for investigation of protein-protein interaction networks","volume":"230","author":"Iacobucci","year":"2021","journal-title":"J. Proteom."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"4007","DOI":"10.1016\/S0021-9258(19)41496-8","article-title":"High resolution two-dimensional electrophoresis of proteins","volume":"250","year":"1975","journal-title":"J. Biol. Chem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1007\/978-1-61779-821-4_27","article-title":"Two-dimensional blue native\/SDS gel electrophoresis of multiprotein complexes","volume":"869","author":"Lasserre","year":"2012","journal-title":"Methods Mol. Biol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"220","DOI":"10.1006\/abio.1994.1112","article-title":"Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis","volume":"217","author":"Cramer","year":"1994","journal-title":"Anal. Biochem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"418","DOI":"10.1038\/nprot.2006.62","article-title":"Blue native page","volume":"1","author":"Wittig","year":"2006","journal-title":"Nat. Protoc."},{"key":"ref_35","first-page":"e2164","article-title":"Blue native polyacrylamide gel electrophoresis (bn-page) for analysis of multiprotein complexes from cellular lysates","volume":"24","author":"Fiala","year":"2011","journal-title":"J. Vis. Exp."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1002\/pmic.200700205","article-title":"How to analyze protein complexes by 2d blue native sds-page","volume":"7","author":"Reisinger","year":"2007","journal-title":"Proteomics"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1016\/0003-2697(91)90094-A","article-title":"Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form","volume":"199","year":"1991","journal-title":"Anal. Biochem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1327","DOI":"10.1002\/elps.200305844","article-title":"Blue silver: A very sensitive colloidal coomassie g-250 staining for proteome analysis","volume":"25","author":"Candiano","year":"2004","journal-title":"Electrophoresis"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"627","DOI":"10.1586\/epr.12.63","article-title":"A blue dive: From \u2019blue fingers\u2019 to \u2019blue silver\u2019: A comparative overview of staining methods for in-gel proteomics","volume":"9","author":"Panfoli","year":"2012","journal-title":"Expert Rev. Proteom."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1002\/elps.1150080203","article-title":"Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels","volume":"8","author":"Blum","year":"1987","journal-title":"Electrophoresis"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"601","DOI":"10.1002\/(SICI)1522-2683(19990301)20:3<601::AID-ELPS601>3.0.CO;2-6","article-title":"Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: A method for the removal of silver ions to enhance sensitivity","volume":"20","author":"Gharahdaghi","year":"1999","journal-title":"Electrophoresis"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1016\/j.toxicon.2014.04.012","article-title":"Snake venom glutaminyl cyclases: Purification, cloning, kinetic study, recombinant expression, and comparison with the human enzyme","volume":"86","author":"Wang","year":"2014","journal-title":"Toxicon"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"6549","DOI":"10.1021\/acs.jmedchem.1c00325","article-title":"Glutaminyl cyclase, diseases, and development of glutaminyl cyclase inhibitors","volume":"64","author":"Xu","year":"2021","journal-title":"J. Med. Chem."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"10635","DOI":"10.1074\/jbc.271.18.10635","article-title":"Complete amino acid sequence and identification of the platelet glycoprotein ib-binding site of jararaca gpib-bp, a snake venom protein isolated from bothrops jararaca","volume":"271","author":"Kawasaki","year":"1996","journal-title":"J. Biol. Chem."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"538","DOI":"10.1111\/jth.13617","article-title":"Mutant botrocetin-2 inhibits von willebrand factor-induced platelet agglutination","volume":"15","author":"Matsui","year":"2017","journal-title":"J. Thromb. Haemost."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"960","DOI":"10.1111\/jth.13987","article-title":"Functional characterization of recombinant snake venom rhodocytin: Rhodocytin mutant blocks clec-2\/podoplanin-dependent platelet aggregation and lung metastasis","volume":"16","author":"Sasaki","year":"2018","journal-title":"J. Thromb. Haemost."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"13576","DOI":"10.1074\/jbc.272.21.13576","article-title":"Platelet activation and signal transduction by convulxin, a c-type lectin from crotalus durissus terrificus (tropical rattlesnake) venom via the p62\/gpvi collagen receptor","volume":"272","author":"Clemetson","year":"1997","journal-title":"J. Biol. Chem."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1189","DOI":"10.1073\/pnas.94.4.1189","article-title":"Cloning and sequence analysis of a bothrops jararaca cdna encoding a precursorof seven bradykinin-potentiating peptides and a c-type natriuretic peptide","volume":"94","author":"Murayama","year":"1997","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Oguiura, N., Correa, P.G., Rosmino, I.L., de Souza, A.O., and Pasqualoto, K.F.M. (2021). Antimicrobial activity of snake beta-defensins and derived peptides. Toxins, 14.","DOI":"10.3390\/toxins14010001"},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Lomate, P.R., Jadhav, B.R., Giri, A.P., and Hivrale, V.K. (2013). Alterations in the helicoverpa armigera midgut digestive physiology after ingestion of pigeon pea inducible leucine aminopeptidase. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0074889"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1016\/0041-0101(91)90106-2","article-title":"Comparative study of nine bothrops snake venoms from adult female snakes and their offspring","volume":"29","author":"Furtado","year":"1991","journal-title":"Toxicon"},{"key":"ref_52","doi-asserted-by":"crossref","unstructured":"Santoro, M.L., do Carmo, T., Cunha, B.H., Alves, A.F., Zelanis, A., Serrano, S.M., Grego, K.F., Sant\u2019Anna, S.S., Barbaro, K.C., and Fernandes, W. (2015). Ontogenetic variation in biological activities of venoms from hybrids between bothrops erythromelas and bothrops neuwiedi snakes. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0145516"},{"key":"ref_53","doi-asserted-by":"crossref","unstructured":"Lotto, N.P., de Albuquerque Modesto, J.C., Sant\u2019Anna, S.S., Grego, K.F., Guarnieri, M.C., Lira-da-Silva, R.M., Santoro, M.L., and Oguiura, N. (2021). The absence of thrombin-like activity in bothrops erythromelas venom is due to the deletion of the snake venom thrombin-like enzyme gene. PLoS ONE, 16.","DOI":"10.1371\/journal.pone.0248901"},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"1363","DOI":"10.1111\/j.1538-7836.2008.02995.x","article-title":"Bothrops protease a, a unique highly glycosylated serine proteinase, is a potent, specific fibrinogenolytic agent","volume":"6","author":"Prezoto","year":"2008","journal-title":"J. Thromb. Haemost."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"597","DOI":"10.1042\/bj0680597","article-title":"The proteolytic activity of the venom of bothrops jararaca","volume":"68","author":"Henriques","year":"1958","journal-title":"Biochem. J."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"595186","DOI":"10.1155\/2014\/595186","article-title":"Isolation and biochemical characterization of a new thrombin-like serine protease from bothrops pirajai snake venom","volume":"2014","author":"Zaqueo","year":"2014","journal-title":"Biomed. Res. Int."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1016\/j.cbpa.2016.01.021","article-title":"Bbrzsp-32, the first serine protease isolated from bothrops brazili venom: Purification and characterization","volume":"195","author":"Zaqueo","year":"2016","journal-title":"Comp. Biochem. Physiol. A Mol. Integr. Physiol."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1016\/j.toxicon.2016.11.001","article-title":"Serine proteinases from bothrops snake venom activates pi3k\/akt mediated angiogenesis","volume":"124","author":"Bhat","year":"2016","journal-title":"Toxicon"},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"1149","DOI":"10.1016\/0041-0101(96)00057-8","article-title":"An aqueous endpoint assay of snake venom phospholipase a2","volume":"34","author":"Holzer","year":"1996","journal-title":"Toxicon"},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"411","DOI":"10.1016\/0041-0101(88)90010-4","article-title":"An alternative in vitro method for testing the potency of the polyvalent antivenom produced in costa rica","volume":"26","author":"Avila","year":"1988","journal-title":"Toxicon"},{"key":"ref_61","doi-asserted-by":"crossref","unstructured":"Thomazini, C.M., Sachetto, A.T.A., Albuquerque, C.Z., Mattaraia, V.G.M., de Oliveira, A.K., Serrano, S.M.T., Lebrun, I., Barbaro, K.C., and Santoro, M.L. (2021). Involvement of von willebrand factor and botrocetin in the thrombocytopenia induced by bothrops jararaca snake venom. PLoS Negl. Trop. Dis., 15.","DOI":"10.1371\/journal.pntd.0009715"},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"982","DOI":"10.1016\/j.toxicon.2007.01.009","article-title":"Enzymatic and immunochemical characterization of bothrops insularis venom and its neutralization by polyspecific bothrops antivenom","volume":"49","author":"Lira","year":"2007","journal-title":"Toxicon"},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1007\/978-1-4939-6412-3_2","article-title":"Gel-filtration chromatography","volume":"1485","author":"Cummins","year":"2017","journal-title":"Methods Mol. Biol."},{"key":"ref_64","doi-asserted-by":"crossref","first-page":"5325","DOI":"10.1007\/s00018-021-03856-0","article-title":"Recent progress in mass spectrometry-based strategies for elucidating protein-protein interactions","volume":"78","author":"Low","year":"2021","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"e2100190","DOI":"10.1002\/pmic.202100190","article-title":"Protein-protein interaction networks as miners of biological discovery","volume":"22","author":"Wang","year":"2022","journal-title":"Proteomics"},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"680","DOI":"10.1038\/227680a0","article-title":"Cleavage of structural proteins during the assembly of the head of bacteriophage t4","volume":"227","author":"Laemmli","year":"1970","journal-title":"Nature"},{"key":"ref_67","doi-asserted-by":"crossref","first-page":"304","DOI":"10.1016\/j.toxicon.2005.11.007","article-title":"Sex-based individual variation of snake venom proteome among eighteen bothrops jararaca siblings","volume":"47","author":"Menezes","year":"2006","journal-title":"Toxicon"},{"key":"ref_68","doi-asserted-by":"crossref","first-page":"2476","DOI":"10.1002\/elps.200500047","article-title":"Detection and quantification of leucyl aminopeptidase after native electrophoresis using leucine-p-nitroanilide","volume":"26","year":"2005","journal-title":"Electrophoresis"},{"key":"ref_69","doi-asserted-by":"crossref","first-page":"76","DOI":"10.1016\/j.thromres.2019.01.018","article-title":"Optimization of von willebrand factor multimer analysis in vertical mini-gel electrophoresis systems: A rapid procedure","volume":"175","author":"Thomazini","year":"2019","journal-title":"Thromb. Res."},{"key":"ref_70","doi-asserted-by":"crossref","first-page":"385","DOI":"10.2144\/97233bm08","article-title":"Enhancement of diaminobenzidine colorimetric signal in immunoblotting","volume":"23","author":"Pukac","year":"1997","journal-title":"Biotechniques"},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1016\/j.jim.2003.10.005","article-title":"Simultaneous isolation of platelet factor 4 and glycoprotein iib-iiia complex from rabbit platelets, and characterization of specific chicken antibodies to assay them","volume":"284","author":"Santoro","year":"2004","journal-title":"J. Immunol. Methods"},{"key":"ref_72","doi-asserted-by":"crossref","first-page":"2856","DOI":"10.1038\/nprot.2006.468","article-title":"In-gel digestion for mass spectrometric characterization of proteins and proteomes","volume":"1","author":"Shevchenko","year":"2006","journal-title":"Nat. Protoc."}],"container-title":["Toxins"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6651\/14\/10\/661\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T00:38:08Z","timestamp":1760143088000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6651\/14\/10\/661"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,9,23]]},"references-count":72,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2022,10]]}},"alternative-id":["toxins14100661"],"URL":"https:\/\/doi.org\/10.3390\/toxins14100661","relation":{},"ISSN":["2072-6651"],"issn-type":[{"value":"2072-6651","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,9,23]]}}}