Mouse anti hsp90α β

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Mouse anti-HSP90α/β is a primary antibody that binds to the heat shock protein 90 alpha and beta isoforms. It is used in research applications to detect and quantify the presence of these proteins.

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Hsp90α (2 citations)

4 protocols using mouse anti hsp90α β

Western Blot Analysis of Ubiquitin Regulators

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A total of 10–50 μg protein was extracted from cells and used for Western blot analysis. Briefly, the cells were lysed using protein lysis buffer (50 mM Tris–Cl, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 200 mM Na3VO4, 1x proteinase inhibitor, pH 7.4) and the protein concentration was measured using a Micro BCA^TM Protein Assay kit (Thermo Scientific), based on the standard curve of BSA. The antibodies used for immunoblot analysis were purchased and assessed for equal loading as follows: rabbit anti-USP7 (#A300-034, dilution ratio 1:1000) and rabbit anti-USP47 (#A301-048A, 1:1000) from Bethyl Laboratories; mouse anti-Flag (#F1804, 1:1000) from Sigma Aldrich; rabbit anti-ubiquitin (#3933, 1:2000) from Cell Signaling; mouse anti-p53 (#sc-126, 1:1000), and mouse anti-HSP90α/β (#sc-13119, 1:5000) from Santa Cruz Biotechnology; rabbit anti-β-actin (#LF-PA0207, 1:5000) from AbFrontier. Samples were analyzed using SDS-PAGE, and chemiluminescence was measured using the Ez-Capture MG imaging system (ATTO Corporation).

Shin S.C., Park J., Kim K.H., Yoon J.M., Cho J., Ha B.H., Oh Y., Choo H., Song E.J, & Kim E.E. (2023). Structural and functional characterization of USP47 reveals a hot spot for inhibitor design. Communications Biology, 6, 970.

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Western Blot Analysis of Protein Extracts

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Western blot analyses on 10–50 μg protein extracts from the cells were performed as described earlier. The cells were lysed using a protein lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Na₄P₂O₇, 50 mM NaF, 5 mM β-glycerophosphate, 1 mM Na₃VO₄) containing protease inhibitor (Roche, Basel, Switzerland). The lysates were quantified by a Micro BCA^TM Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA) based on the standard curve using BSA. The following antibodies were used for immunoblot analysis: rabbit anti-USP47 (A301-048A, Bethyl Laboratories, Montgomery, TX, USA), rabbit anti-RPL11 (ab79352, Abcam), mouse anti-p53 (sc-126, Santa Cruz, Dallas, TX, USA), mouse anti-MDM2 (PA5-11353, Invitrogen), anti-p21 (2947S, Cell signaling Technology, Danvers, MA, USA), mouse anti-XIAP (sc-55551, Santa Cruz), mouse anti-caspase 3 (sc-271028, Santa Cruz), mouse anti-HSP90α/β (sc-13119, Santa Cruz), rabbit anti-β-actin (LF-PA0207, Ab frontier, Seoul, Korea), mouse anti-HA (sc-7392, Santa Cruz), and mouse anti-Flag (F1804, Sigma-Aldrich). The band intensity was quantified using image J and normalized by an indicated loading control protein. Original images of the western blot can be found in Supplementary File S1.

Park J., Cho M., Cho J., Kim E.E, & Song E.J. (2022). MicroRNA-101-3p Suppresses Cancer Cell Growth by Inhibiting the USP47-Induced Deubiquitination of RPL11. Cancers, 14(4), 964.

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Western Blot Analysis of Hedgehog Pathway

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Cells were lysed in cold RIPA buffer (50mM Tris-HCl pH 7.5, 1% NP-40, 150mM NaCl, 5mM EDTA, 0.25% NaDOC, and 0.1% SDS) supplemented with protease and phosphatase inhibitors and centrifuged at 20,000× g for 20 min at 4°C [26 (link)]. Supernatant was collected as whole cell extract (WCE). Equal amounts of protein were resolved by SDS-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, and incubated for 1 h in blocking buffer at room temperature. The following primary antibodies were used: mouse anti-GLI1 (#2643, Cell Signaling Technologies, Danver, MA, USA), mouse anti-SMO (sc-166685, Santa Cruz Biotechnology, Dallas, TX, USA), and mouse anti-HSP90α/β (sc-13119, Santa Cruz Biotechnology, Dallas, TX, USA). After incubation with the corresponding horseradish peroxidase (HRP)-coupled secondary antibody, membranes were developed by using SuperSignal West Femto (Thermo Fisher Scientific, Waltham, MA, USA) and imaged with ChemiDoc Imaging Systems (Bio-Rad, Hercules, CA, USA).

Pietrobono S., Franci L., Imperatore F., Zanini C., Stecca B, & Chiariello M. (2021). MAPK15 Controls Hedgehog Signaling in Medulloblastoma Cells by Regulating Primary Ciliogenesis. Cancers, 13(19), 4903.

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Western Blot Analysis of Mitochondrial Proteins

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Tissues were lysed in modified RIPA buffer (450 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% Deoxycholic acid (sodium salt), 50 mM Tris pH 7.5, 1x complete protease inhibitor). Total cellular protein (30 μg) was separated on SDS-PAGE gels and transferred to nitrocellulose membrane. After blocking in 5% defatted milk, membranes were incubated in primary antibody at 4°C overnight. The primary antibodies used in this study were: rabbit anti-UCP1 (Abcam, ab10983, 1:40,000 for BAT, 1:2000 for co-IP experiments), rabbit anti-EMRE (Santa Cruz Biotech, sc-86337, 1:300), rabbit anti-MCU (Cell Signaling Technologies, 14997, 1:2000) , rabbit anti-MICU1 (Sigma, HPA037479, 1:1000), rabbit anti-HA (Cell Signaling Technologies, 3724, 1:2000) , mouse anti-SDHB (Santa Cruz Biotech, sc-271548, 1:2000) , mouse anti-FLAG (Abmart, 314375, 1:5000), mouse anti-PDHE1α (Santa Cruz Biotech, sc-377092, 1:5000), rabbit anti-phospho-PDHE1α S293 (Millipore, ABS204, 1:2000), rabbit anti-AKT(pS473) (Cell Signaling Technologies, 9271, 1:2000) , rabbit anti-AKT (Cell Signaling Technologies, 9272, 1:2000) , mouse anti-HSP90α/β (Santa Cruz Biotech, sc13119, 1:10000), mouse anti-NDUFS1 (Abcam, ab22094, 1:10000). The membranes were then incubated in HRP-conjugated secondary antibodies at 37°C for 1 h and visualized using Tanon 5200 Chemiluminescent Imaging System.

Xue K., Wu D., Wang Y., Zhao Y., Shen H., Yao J., Huang X., Li X., Zhou Z., Wang Z, & Qiu Y. (2022). The mitochondrial calcium uniporter engages UCP1 to form a thermoporter that promotes thermogenesis. Cell metabolism, 34(9).

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