Co-immunoprecipitation protocol was adapted from Hofmann et al. [33 (link)]. Briefly, 10 × 106 cells were treated with 20 µM of MTI-101 for 10 min in Live Cell Imaging Solution (Molecular Probes by Life Technologies), centrifuged at 4 °C and lysed with 1% Triton X-100 (Thermo Scientific; 85111) in PBS, with protease and phosphatase inhibitors cocktail (Millipore Sigma). BCA assay was used to quantify protein concentration. 1 mg of protein for each treatment and control groups were incubated with the primary antibody (TRPC1; Santa Cruz Biotechnology, and host species IgG) overnight rotating in 4 °C. Later, protein A/G plus agarose (Santa Cruz Biotechnology; sc-2003) was added to the sample and allowed to incubate rotating in 4 °C for one hour. Samples were washed 3 times in lysis buffer and samples were subjected to Western blot analysis.
Trpc1
Manufactured by Santa Cruz Biotechnology
Sourced in United States
TRPC1 is a membrane protein that functions as a calcium-permeable cation channel. It is involved in the regulation of cellular calcium homeostasis and signaling processes.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)
Anti mouse horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Stim1, by Santa Cruz Biotechnology (1 mentions)
Orai2, by Santa Cruz Biotechnology (1 mentions)
Orai1, by Santa Cruz Biotechnology (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Thapsigargin, by Thermo Fisher Scientific (1 mentions)
Alpha smooth muscle actin, by Abcam (1 mentions)
Cytokeratin 8, by Abcam (1 mentions)
Cytokeratin 18, by Abcam (1 mentions)
Hsc70, by Santa Cruz Biotechnology (1 mentions)
Stim1, by Abcam (1 mentions)
Orai1, by Abcam (1 mentions)
D luciferin, by Merck Group (1 mentions)
Cyclopiazonic acid, by Merck Group (1 mentions)
Matrigel, by Corning (1 mentions)
3 protocols using trpc1
1
Co-immunoprecipitation of TRPC1 Protein
2
Western Blot Analysis of Lung Proteins
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Total proteins from frozen left lung samples were isolated using a RIPA lysis buffer
(Thermo Fisher) supplemented with a Protease inhibitor tablet cocktail according to
manufacturer's instructions (Pierce™, Thermo Fisher). Protein concentrations were
calculated using DC Protein Assay (Bio-Rad Hercules, CA, USA) following the manufacture
instructions. Isolated proteins were stored at −80°C until use. Ten micrograms of protein
were resolved in an SDS-PAGE (10–12% gradient gels) followed by transferring of proteins
onto nitrocellulose membranes. After appropriate blocking (5% nonfat dried milk) the blots
were probed with primary antibodies for TRPC1 (Santa Cruz, Dallas, TX, USA, sc-133076),
TRPC6 (Santa Cruz, sc-515837), STIM1 (Santa Cruz, sc-166840), ORAI2 (Santa Cruz,
sc-376757), and ORAI1 (Santa Cruz, sc-377281), diluted 1:1000 in 5% powdered nonfat dry
milk overnight at 4°C. After washing, membranes were incubated with horseradish
peroxidase-conjugated secondary anti-mouse antibody (Jackson ImmunoResearch, West Grove,
PA, USA) diluted 1:5000 in 5% powdered nonfat dry milk. Levels of proteins were normalized
to the β-actin (Sigma-Aldrich, AC-74) content of the same sample. The signals obtained
were scanned with densitometry through a detection device by chemiluminescence (Odyssey
Imaging System LI-COR Biosciences, Lincoln, NE, USA) quantified with the Image J software
(NIH, Bethesda, MD, USA).
(Thermo Fisher) supplemented with a Protease inhibitor tablet cocktail according to
manufacturer's instructions (Pierce™, Thermo Fisher). Protein concentrations were
calculated using DC Protein Assay (Bio-Rad Hercules, CA, USA) following the manufacture
instructions. Isolated proteins were stored at −80°C until use. Ten micrograms of protein
were resolved in an SDS-PAGE (10–12% gradient gels) followed by transferring of proteins
onto nitrocellulose membranes. After appropriate blocking (5% nonfat dried milk) the blots
were probed with primary antibodies for TRPC1 (Santa Cruz, Dallas, TX, USA, sc-133076),
TRPC6 (Santa Cruz, sc-515837), STIM1 (Santa Cruz, sc-166840), ORAI2 (Santa Cruz,
sc-376757), and ORAI1 (Santa Cruz, sc-377281), diluted 1:1000 in 5% powdered nonfat dry
milk overnight at 4°C. After washing, membranes were incubated with horseradish
peroxidase-conjugated secondary anti-mouse antibody (Jackson ImmunoResearch, West Grove,
PA, USA) diluted 1:5000 in 5% powdered nonfat dry milk. Levels of proteins were normalized
to the β-actin (Sigma-Aldrich, AC-74) content of the same sample. The signals obtained
were scanned with densitometry through a detection device by chemiluminescence (Odyssey
Imaging System LI-COR Biosciences, Lincoln, NE, USA) quantified with the Image J software
(NIH, Bethesda, MD, USA).
3
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.
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