Polyclonal antibodies against human proteasome subunits PSMA3, PSMA5, and PSMB5статья
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Web of Science ,
Scopus
Статья опубликована в журнале из списка Web of Science и/или Scopus
Дата последнего поиска статьи во внешних источниках: 1 апреля 2017 г.
Авторы:
Livinskaya V.A.a ,
Ivanov V.A.a ,
Fedorova O.A.a ,
Vorontsova D.N.a,
Barlev N.A.a.b ,
Nikiforov A.A.a.c
Журнал:
Hybridoma
Том:
31
Номер:
4
Год издания:
2012
Издательство:
Mary Ann Liebert Inc.
Местоположение издательства:
United States
Первая страница:
272
Последняя страница:
278
DOI:
10.1089/hyb.2012.0004
Аннотация:
A proteasome is a multi-subunit protein complex, which plays a central role in ubiquitin-dependent protein degradation in all eukaryotic cells. The 26S proteasome is composed of a catalytic 20S core complex and one or two 19S regulatory complexes. The 20S core complex forms a cylinder consisting of four stacked rings of seven α (PSMA1-7) or β (PSMB1-7) subunits. Target proteins are degraded in the cavity of the 20S complex due to proteolytic activities of three β subunits having catalytic sites located on the inner surface of the cylinder. The aim of this study was the generation of polyclonal antibodies against human proteasome subunits PSMA3, PSMA5, and PSMB5 and characterization of their experimental applications. To construct GST-fusion proteins, DNA sequences encoding PSMA3, PSMA5, and PSMB5 were cloned into prokaryotic expression vectors pGEX-5X-1 or pGEX-4T-3. Recombinant proteins GST-PSMA3, GST-PSMA5, and GST-PSMB5 were highly expressed in E. coli BL21 (DE3) cells, purified by glutathione-affinity chromatography and further used for rabbit immunization. The activity and specificity of the obtained antibody-containing sera were evaluated using Western blot analysis and immunoprecipitation. We have shown by Western blot analysis that our anti-PSMA3, anti-PSMA5, and anti-PSMB5 antibodies recognized both recombinant and endogenous proteins from different human cell lines. We have also shown that anti-PSMA3 and anti-PSMA5 sera were able to recognize and immunoprecipitate native forms of both endogenous and overexpressed FLAG-tagged proteins PSMA3 and PSMA5, respectively. Thus, the antibodies generated against PSMA3, PSMA5, and PSMB5 can be used in various experimental applications, including the evaluation of cellular levels of proteasome subunits in cell extracts and affinity purification of the endogenous and/or overexpressed proteasome subunits, which facilitates subsequent analysis of their post-translational modifications as well as protein-protein interactions in vivo. © 2012 Copyright, Mary Ann Liebert, Inc.
Добавил в систему:
Никифоров Андрей Анатольевич