Protein isolation was performed on 15 × 104 cells seeded on 6-well plate by cOmplete Lysis-M, EDTA-free (Roche) according to the manufacturer’s instructions. Protein extracts, separated by SDS-PAGE were transferred onto PVDF membranes, then, incubated with antibodies targeted against STIM1 (3 μg/μl, Abcam), Orai1 (1:750, Abcam) and β-actin (1:5000, Sigma) overnight at 4 °C. Membranes were incubated with secondary antibodies (1:5000, anti-rabbit or anti-mouse, LI-COR) for 1 h via shaking at room temperature. Protein bands were visualized in an infrared imager (Odyssey, LI-COR) based on the appropriate channel properties (680RD or 800CW) of secondary antibodies.
Orai1
Manufactured by Abcam
Sourced in United States
Orai1 is a calcium release-activated calcium (CRAC) channel protein that plays a key role in the regulation of calcium homeostasis. It is responsible for the influx of calcium into cells in response to depletion of calcium from the endoplasmic reticulum. Orai1 is essential for various cellular processes, including immune function, cell growth, and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (4 mentions)
St505, by Beyotime (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Stim1, by Abcam (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
Complete lysis m edta free, by Roche (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Biospectrum imaging system, by Analytik Jena (1 mentions)
Anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Pro prep lysis buffer, by iNtRON Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Thiamet g, by Bio-Techne (1 mentions)
Skf96365, by Bio-Techne (1 mentions)
Pugnac, by Abcam (1 mentions)
O glcnac, by Abcam (1 mentions)
Mg132, by Cell Signaling Technology (1 mentions)
2 apb, by Bio-Techne (1 mentions)
Thapsigargin, by Thermo Fisher Scientific (1 mentions)
6 protocols using orai1
1
Western Blot Analysis of STIM1 and Orai1
2
Orai1 Expression in CCRCC
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Western blot analysis was performed on 8 paired fresh tissue including CCRCC tissue and adjacent non-tumor renal tissue. Paired fresh tissues were lysed using 2 mL of PRO-PREP lysis buffer (iNtRon Biotechnology, Daejeon, Korea), and then ground for 15-20 seconds on ice using a homogenizer (ProScience, Woburn, MA, USA). Equal amount of protein lysates used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the immobilon-P membrane (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk and incubated with primary antibodies, including Orai1 (Abcam) and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4°C. The membranes were then incubated with secondary antibodies, including Anti-Rabbit IgG (HRP) (Santa Cruz Biotechnology) and Anti-mouse IgG (Santa Cruz Biotechnology) at room temperature for an hour. Immunoblotting bands were detected and quantified using Biospectrum Imaging System (UVP, Upland, CA, USA) and ImageJ software (available at http://rsb.info.nih.gov/ij/index.html ).
3
Quantitative Western Blot Analysis
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Lung tissues or hBSMCs were lysed in RIPA buffer containing phosphatase and protease inhibitors (ST505, Beyotime, China) on ice for 30 min and centrifuged at 12,000 rpm for 20 min at 4°C. Protein concentrations were detected using a BCA protein assay kit (Beyotime, China), and equal amounts of protein (40 μg each lane) were separated with SDS-PAGE and then transferred to Poly (vinylidene fluoride) (PVDF) membranes (Millipore, USA). Membranes were incubated with primary antibody of Orai1 (rabbit anti-human, 1:4000, Abcam, USA) and β-actin (goat anti-rabbit, 1:5000, Sigma, St. Louis, MO, USA) for 18 h, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature. Enhanced chemiluminescence (ECL) was used to identify the immunoreactive bands, and blots densitometry was analyzed by ImageJ software.
4
Investigating TRPM7 and O-GlcNAcylation
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2-APB, SKF96365, and thiamet G were obtained from Tocris Bioscience (Bristol, UK). PugNAc and antibodies for TRPM7, ORAI1, STIM1, OGA, O-GlcNAc and ITGB7 were obtained from Abcam (Cambridge, UK). Antibody for ubiquitin was from Santa Cruz Biotechnology (Dallas, TX, USA) and secondary antibodies were from EMD Millipore (Berlington, MA, USA). MG-132 and all other antibodies were from Cell Signaling Technology (Beverly, MA, USA). Other reagents were from Sigma-Aldrich (Dallas, MA, USA).
5
Antibody Screening for Immunoblotting and Immunohistochemistry
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Antibodies used for immunoblotting were as follows: HSC70 (# sc-7298) and TRPC1 (# sc-133076) from Santa Cruz Biotechnology; STIM1 (# ab57834) from Abcam; Orai1 (# O8264) and beta-actin (# SAB1305567) from Millipore-Sigma; Caveolain-1 (#610057) from BD Biosciences. In-house generated Protein G-purified rabbit polyclonal rabbit anti-EHD2 antisera has been described previousl (George et al., 2007 (link)). The horseradish peroxidase (HRP)-conjugated Protein A or HRP-conjugated rabbit anti-mouse secondary antibody for immunoblotting were from Invitrogen. The alpha smooth muscle actin (# ab7817), cytokeratin 8 (#53280), cytokeratin 18 (# 133263), Ki67 (# ab16667) antibodies for immunohistochemistry (IHC) and immunofluorescence (IF) staining were from Abcam. Thapsigargin (# T7459) and Fluo 4AM (# 14201) were from Thermo Fisher Scientific. Cyclopiazonic acid (# C1530) and D-luciferin (#L9504) were from Millipore Sigma. SKF96365 (cat. # S7999), SOCE inhibitor CM4620 (# S6834) from SelleckChem, Matrigel (# 356230) from Corning, and Isoflurane (# 502017) from MWI Animal Health.
6
Orai1 Protein Expression in Lung Tissues
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Lung tissues or hBSMCs were lysed in RIPA buffer containing phosphatase and protease inhibitors (ST505, Beyotime, China) on ice for 30 min and centrifuged at 12,000 rpm for 20 min at 4°C. Protein concentrations were detected using a BCA protein assay kit (Beyotime, China), and equal amounts of protein (40 µg each lane) were subjected to SDS-PAGE and then transferred to Poly (vinylidene uoride) (PVDF) membranes (Millipore, USA). Membranes were incubated by primary antibody of Orai1 (rabbit anti human, 1:4000, Abcam, USA) and β-actin (goat anti rabbit, 1:5000, Sigma, St. Louis, MO, USA) for 18 h, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature. enhanced chemiluminescence (ECL) was used to detect the immunoreactive bands, and blots densitometry was analyzed by ImageJ software.
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