For tissue distribution of mRNA and protein expressions, data on the target genes were obtained from “The Tissue Atlas” category of “The Human Protein Atlas/HPA” (http://www.proteinatlas.org/ ) [51 (link)]. The mRNA expression from the “The Genotype-Tissue Expression/GTEx Dataset” was chosen for demonstration with reference to the normalized consensus dataset Immunohistochemistry staining was performed on normal human tissue samples. AR expression was detected with a rabbit antibody (1:250, HPA004733, Sigma-Aldrich) and validated with another rabbit antibody (1:500, GTX62599, GeneTex). ACE2 expression was primarily detected with a rabbit antibody (1:250, HPA000288, Sigma-Aldrich) and confirmed with a mouse antibody (1:5000, CAB026174, R&D Systems); TMPRSS2 expression was detected with a rabbit antibody (1:300, HPA035787, Sigma-Aldrich). The information on expression intensity and specific cell types that express respective genes were extracted from the staining reports for each staining type in the database.
Hpa000288
Manufactured by Merck Group
Sourced in United States, United Kingdom
HPA000288 is a laboratory instrument used for analytical measurements. The core function of this product is to facilitate precise data collection and analysis. No further details on its intended use are provided.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Gtx62599, by GeneTex (1 mentions)
Cab026174, by R&D Systems (1 mentions)
Hpa035787, by Merck Group (1 mentions)
Ab15348, by Abcam (1 mentions)
Ab92323, by Abcam (1 mentions)
Nitrocellulose membrane, by Advansta (1 mentions)
Ab49900, by Abcam (1 mentions)
Complete mini edta free protease inhibitor, by Merck Group (1 mentions)
Eblot l1, by GenScript (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Fusion fx7 imaging system, by Vilber (1 mentions)
Westernbright quantum, by Advansta (1 mentions)
4 protocols using hpa000288
1
Tissue Expression Profiling of AR, ACE2, and TMPRSS2
2
Characterization of Anti-ACE2 and Anti-Pituitary Antibodies
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We used a specific anti-ACE2 rabbit polyclonal antibody (HPA000288; Sigma-Aldrich, St. Louis, MO, USA). The antigen sequence that produces the antibody has 86% homology with the
corresponding region of bovine ACE2, as shown by the green highlighted region inSupplementary Fig. 1 . In particular, ACE2 has a
single transmembrane region [18 (link)], as indicated by the blue highlighted region inSupplementary
Fig. 1 . There is 91% homology between the extracellular regions of bovine and human ACE2 (the NCBI reference sequences of bovine and human ACE2 are NP_001019673.2 and BAB40370,
respectively).
We also used a guinea pig polyclonal antibody that recognizes the N-terminal extracellular domain of the bovine GnRH receptor (anti-GnRHR). The specificity of the anti-GnRHR antibody has
been verified previously [16 (link)]. We used a mouse monoclonal anti-LH antibody (clone 518-B7) [19 (link)] and a mouse
monoclonal anti-FSH antibody (clone A3C12) [20 (link)] for immunohistochemical analysis of AP tissue and cultured AP cells. These antibodies do not
cross-react with other pituitary hormones [20 (link), 21 (link)].
corresponding region of bovine ACE2, as shown by the green highlighted region in
single transmembrane region [18 (link)], as indicated by the blue highlighted region in
Fig. 1
respectively).
We also used a guinea pig polyclonal antibody that recognizes the N-terminal extracellular domain of the bovine GnRH receptor (anti-GnRHR). The specificity of the anti-GnRHR antibody has
been verified previously [16 (link)]. We used a mouse monoclonal anti-LH antibody (clone 518-B7) [19 (link)] and a mouse
monoclonal anti-FSH antibody (clone A3C12) [20 (link)] for immunohistochemical analysis of AP tissue and cultured AP cells. These antibodies do not
cross-react with other pituitary hormones [20 (link), 21 (link)].
3
Protein Expression and Quantification
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Cells were lysed using RIPA buffer (Thermo Fisher Scientific) containing cOmplete, Mini, EDTA-free protease inhibitor (Sigma-Aldrich). Protein concentration was determined by BCA Protein Assay kit (Thermo Fisher Scientific). 10 µg/sample with 4x Laemmli Buffer (Bio-Rad, Hercules, CA, USA) containing β-mercaptoethanol was resolved in 20% Mini-PROTEAN TGX Gel (Witec AG, Sursee, Switzerland). PNGase treatments have been performed according to the manufacturer’s protocol (New England BioLabs, Frankfurt, Germany). Gels were transblotted on nitrocellulose membrane (Advansta) using eBlot L1 (GenScript, Piscataway, NJ, USA). Membranes were blocked and stained with primary antibody overnight in 5% nonfat dry milk in 0.1% phosphate-buffered saline Tween, visualized with horseradish peroxidase (HRP)-conjugated secondary antibody using WesternBright Quantum (Advansta Corporation, Menlo Park, CA, USA) and imaged by Fusion FX 7 Imaging System (Vilber Lourmat, Eberhardzell, Germany). Antibodies used are rabbit anti-ACE2 (1:500, ab15348, Abcam, Cambridge, UK), rabbit anti-ACE2 (1:1000, HPA000288, Sigma-Aldrich), rabbit anti-TMPRSS2 (1:1000, ab92323, Abcam), rabbit anti-NRP1 (1:50, HPA030278, Sigma-Aldrich), HRP-conjugated anti-rabbit (1:10000, 11-035-003, Jackson), and HRP-conjugated anti β-actin (1:25000, ab49900, Abcam).
4
ACE2 Protein Expression Across Human Tissues
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Immunohistochemical localization of human ACE2 was evaluated from immunostained specimens provided by Protein Expression Atlas (https://www.proteinatlas.org/ ) [42 (link)]. The dataset included three specimens of duodenum, three specimens of kidney, three specimens of testis, three specimens of lung, and two specimens of nasopharynx. The images of the Fig 2 represent duodenum from 77-years-old female, kidney from 36-years-old male, testis from 38-years-old male, lung from 61-years-old female, and nasopharyngeal mucosa from 78-years-old female. According to Protein Expression Atlas the immunostainings were performed with the rabbit anti-human polyclonal antibody (HPA000288; Sigma Aldrich, St. Louis, MO) raised against 111 N-terminal amino acids of ACE2 and diluted 1:250 for the staining.
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