Human p-RTKs were assayed using Human Phospho-RTK Array Kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. Briefly, p-RTK array membranes were blocked with 5% BSA/TBS (0.01 M Tris-HCl, pH 7.6) for 1 h and incubated with 2 mL of lysate, which was prepared from cell lines after normalization to ensure equal protein amounts. The membranes were washed 3 times with TBS plus 0.1% v/v Tween-20 for 10 min each and 2 times with TBS alone for 10 min each to remove unbound materials. Then, the membranes were incubated with an HRP-conjugated anti-phospho-tyrosine antibody for 2 h at room temperature. The unbound HRP-conjugated antibody was washed away with TBS plus 0.1% Tween-20. Finally, each array membrane was exposed to X-ray film using a chemiluminescence detection system (PerkinElmer Co.). The immunoreactive bands were analyzed by densitometric scanning (TIc scanner, Shimizu Co., Ltd., Kyoto, Japan).
Chemiluminescence detection system
Manufactured by PerkinElmer
Sourced in United States
The Chemiluminescence detection system is a laboratory instrument designed to measure and analyze chemiluminescent signals. It is used to detect and quantify a wide range of biomolecules and chemical compounds that produce light through a chemiluminescent reaction.
Automatically generated - may contain errors
Lab products found in correlation
Human phospho rtk array kit, by R&D Systems (3 mentions)
Las 3000 instrument, by Fujifilm (2 mentions)
Anti α tubulin, by Merck Group (2 mentions)
Raybio human angiogenesis antibody array, by RayBiotech (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Hrp conjugated anti rabbit igg antibody, by GeneTex (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Anti flag, by Merck Group (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Anti flag m2 affinity gel, by Merck Group (1 mentions)
Anti myc, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by GE Healthcare (1 mentions)
Pdvf membrane, by Bio-Rad (1 mentions)
Phosstop, by Roche (1 mentions)
Hrp conjugated goat anti mouse igg, by Bio-Rad (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Mouse anti actin antibody, by Merck Group (1 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
16 protocols using chemiluminescence detection system
1
Phospho-RTK Array Analysis of Cell Lines
2
Coimmunoprecipitation of Ubiquitin Ligases
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COS7 cells [10] (link), [15] (link) were cultured in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% (v/v) fetal bovine serum and 1% penicillin-streptomycin. Cells were transiently transfected using Lipofectamine 2000 transfection reagent (Invitrogen). 4 µg of pCS2-MT-fih-1 was cotransfected with 4 µg of pCS2-FLAG-mib or -mib2 into COS7 cells. 2 µg of pCS2-MT-trabid C (295–716 aa), pCS2-MT-usp1 (349–772 aa) or pCMV-HA-usp9 was cotransfected with 2 µg of pCS2-FLAG-mib or -mib2 into COS7 cells. Cells were harvested and lysed 24 h after transfection in IONIC buffer (50 mM Tris HCl [pH 7.4], 150 mM NaCl, 1% Triton X-100, containing a protease inhibitor cocktail, Calbiochem). The cell lysate was clarified by centrifugation and incubated with anti-FLAG M2 affinity gel (Sigma) for 2 h at 4°C. The gel was boiled in SDS gel loading buffer and the eluted proteins were electrophoresed on a SDS-PAGE, and transferred to a polyvinylidene difluoride membrane (Millipore). Blots were incubated with primary antibody: anti-HA (Protech), anti-Myc (Santa Cruz) or anti-FLAG (Sigma) for 2 hr. The signal was visualized using a secondary antibody: HRP-conjugated anti-mouse IgG antibody (GeneTex) or HRP-conjugated anti-rabbit IgG antibody (GeneTex), with a chemiluminescence detection system (PerkinElmer Inc).
3
Protein Analysis of Mouse Skin Wound Healing
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Normal mouse skin tissue and mouse wound tissue 7 and 23 days post wounding were lysed in radioimmunoprecipitation (RIPA) buffer supplemented with a phosphatase inhibitor (PhosSTOP, Roche). The lysates were incubated for 20 min on ice and centrifuged at 20,000 × g for 15 min at 4°C. The supernatants were denatured at 95°C for 5 min in SDS sample buffer, consisting of 62.5 mmol/L Tris (pH 6.8), 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, and 0.001% bromophenol blue. Samples were separated by SDS–PAGE, and proteins were transferred onto a PDVF membrane (Bio-Rad, Munich, Germany). Membranes were incubated for 30 min at room temperature in blocking buffer consisting of 5% nonfat dry milk in phosphate-buffered saline (PBS) with 0.05% Tween-20, followed by an appropriate dilution of anti-POSTN Ab (Abcam) or anti-α-tubulin (Sigma) primary antibody overnight at 4°C. Immune complexes were detected with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, Piscataway, NJ), a chemiluminescence detection system (Perkin-Elmer, Waltham, MA), and a LAS-3000 instrument (Fujifilm, Tokyo, Japan)
4
Angiogenesis Cytokine Profiling
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The RayBio Human Angiogenesis Antibody Array (RayBiotech Inc.) was used according to the manufacturer's protocol. This method is a dot-based assay enabling detection and comparison of 20 angiogenesis-specific cytokines. Each array membrane was exposed to an X-ray film using a chemiluminescence detection system (PerkinElmer Co.).
5
Western Blot Analysis of Cellular Proteins
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Cells were lysed with RIPA buffer containing protease and phosphatase inhibitors (Sigma Aldrich Corporation). Lysates were clarified by centrifugation at 14,000× rpm for 15 min at 4°C, subjected to 10% SDS-PAGE and Western blotting using a mouse anti-β-catenin antibody (Becton–Dickinson), a mouse anti-actin antibody (Sigma Aldrich Corporation) or a mouse monoclonal antibody against LGALS3BP (3C12.2). Incubation was performed overnight at 4°C. After washing with PBS containing 0.1% Tween-20, blots were incubated with a goat anti-mouse HRP-conjugated IgG as a secondary antibody (Biorad, Berkeley, CA, USA) at room temperature for 2 h and developed with a chemiluminescence detection system (Perkin-Elmer, Waltham, MA, USA).
6
Immunoblotting Analysis of Signaling Pathways
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Cells were washed twice with ice-cold PBS before the addition of lysis buffer (m-PER Mammalian Protein Extraction reagent, Thermo Scientific, Waltham, MA) and inhibitor cocktail (Sigma, 5μg/mL). Protein concentration was quantified by the Bio-Rad method. 35μg of protein were loaded into each well of a 12.5% SDS-PAGE gel, followed by transfer onto PVDF-membranes (Bio-Rad, Hercules, CA). Membranes were blocked with 5% non-fat dry milk in PBS-T for 1 hour at room temperature. The blots were then incubated with antibodies against the following human proteins: p-mTOR (1:1000; Cell Signaling Technology, Danvers, MA); mTOR (1:1000; Cell Signaling Technology); p-Akt (1:1000; Cell Signaling Technology, Danvers, MA); Akt (1:1000; Cell Signaling Technology); p-S6 ribosomal protein (1:1000; Cell Signaling Technology); S6 ribosomal protein (1:1000; Cell Signaling Technology); caspase-3 (1:1000; Cell Signaling Technology); and actin (1:1000; Cell Signaling Technology) for 1 hour at 4°C. Blots were rinsed 3×10min, then incubated with goat anti-rabbit IgG secondary (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA) for 45 minutes at room temperature. Immunoblots were developed by using the chemiluminescence detection system (PerkinElmer, Waltham, MA), according to the manufacture's protocol, and autoradiography.
7
Apoptosis Pathway Profiling of Telmisartan
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HLF cells were cultured for 12 h after treatment with 100 µM of telmisartan or DMSO control and then lysed in PRO-PREP. The human apoptosis antibody array kit (R&D Systems, Minneapolis, MN, USA) was used to assess the levels of apoptosis-related proteins. Briefly, proteins were captured by antibodies spotted on a nitrocellulose membrane. Then, the levels of apoptosis-related proteins were assessed using an HRP-conjugated antibody followed by detection via chemiluminescence, and each array membrane was exposed to X-ray film using a chemiluminescence detection system (Perkin-Elmer, Waltham, MA, USA). The immunoreactive band density obtained from this array was analyzed by densitometric scanning (TIc scanner; Shimazu Co., Ltd., Kyoto, Japan). We repeated the same experiment three times to compare the telmisartan-treated group with the non-treated group.
8
Angiogenesis Cytokine Profiling
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A Human Angiogenesis Antibody Array (R&D Systems, Minneapolis, MN, USA) was used according to the manufacturer's protocol. This method is a dot-based assay enabling the detection and comparison of 55 angiogenesis-specific cytokines. Each array membrane was exposed to X-ray film using a chemiluminescence detection system (PerkinElmer Co.).
9
Phospho-RTK Array Analysis of Cell Lysates
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Human p-RTKs were assayed using Human Phospho-RTK Array Kits according to the manufacturer's instructions. Briefly, p-RTK array membranes were blocked with 5% BSA/TBS (0.01 M Tris-HCl, pH 7.6) for 1 h and incubated with 2 ml of lysate prepared from the previously mentioned cells after normalization so that the amounts of protein were equal. After 3 washes for 10 min each with TBS plus 0.1% v/v Tween-20 and 2 washes for 10 min with TBS alone to remove unbound materials, the membranes were incubated with an HRP-conjugated anti-phosphotyrosine antibody for 2 h at room temperature. The unbound HRP antibody was washed out with TBS plus 0.1% Tween-20. Finally, each array membrane was exposed to X-ray film using a chemiluminescence detection system (Perkin-Elmer Co.). The density of the immunoreactive band obtained 118 on this array was analyzed by densitometric scanning (TIc 119 scanner; Shimizu Co, Ltd., Kyoto, Japan).
10
Angiogenesis Antibody Array Analysis
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A Human Angiogenesis Antibody Array was used according to the manufacturer's protocol. This method is a dot-based assay enabling the detection and comparison of 55 angiogenesis-specific cytokines. Each array membrane was exposed to X-ray film using a chemiluminescence detection system (Perkin-Elmer Co.). The immunoreactive bands were analyzed by densitometric scanning.
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