Total protein was isolated from CS-1 and SW1353 cells with 1 × RIPA lysis buffer (Upstate Biotechnology, Charlottesville, VA) supplemented with complete protease inhibitor cocktail tablets (Roche Applied Science, IN, USA). Protein concentrations were examined by Protein Assay Reagents (Bio-Rad, Hercules, USA). Equal amounts of denatured proteins were separated by NuPAGE® 4–12% Bis-Tris Gel (Life Technologies) and transferred onto a nitrocellulose membrane (Bio-Rad). After blocking in 5% non-fat milk for two hours, the membranes were incubated with specific primary antibodies WIF1 (Cell Signaling Technology, catalog number: 5652, 1:1000 dilution), Wnt5a/b (Cell Signaling Technology, catalog number: 2530, 1:1000 dilution), LRP6 (Cell Signaling Technology, catalog number: 3395, 1:1000 dilution), Dvl2 (Cell Signaling Technology, catalog number: 3224, 1:1000 dilution), and β-Actin (Sigma-Aldrich, dilution 1:2000) at 4 °C overnight. The membranes were further probed with respective secondary antibodies (LI-COR Biosciences, NE, USA), and scanned by Odyssey® CLx equipment (LI-COR Biosciences, NE, USA) to detect the bands and the density.
Odyssey clx equipment
Manufactured by LI COR
Sourced in United States
The Odyssey® CLx is a high-performance infrared imaging system designed for quantitative Western blotting, fluorescence, and near-infrared (NIR) applications. The system utilizes dual-channel imaging capabilities to detect and quantify multiple targets simultaneously. It features high sensitivity, linear dynamic range, and flexible imaging options to meet the needs of various life science research applications.
Automatically generated - may contain errors
Lab products found in correlation
Protein assay reagent, by Bio-Rad (3 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (3 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
β actin, by Merck Group (2 mentions)
Complete protease inhibitor cocktail tablet, by Roche (2 mentions)
Wnt5a b, by Cell Signaling Technology (1 mentions)
Beckman du 640, by Beckman Coulter (1 mentions)
Mouse monoclonal antibody to human β actin, by Merck Group (1 mentions)
Respective secondary antibodies, by LI COR (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
H3k27me3, by Active Motif (1 mentions)
Cocktail inhibitor, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Enzalutamide, by Cayman Chemical (1 mentions)
Irdye 800cw goat anti rabbit, by LI COR (1 mentions)
Goat anti mouse irdye 680lt secondary antibody, by LI COR (1 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
Spectramax 340pc microplate reader, by Molecular Devices (1 mentions)
9 protocols using odyssey clx equipment
1
Quantifying Wnt Pathway Proteins
2
Western Blotting Procedure for Protein Analysis
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Western blotting reagents and procedures have been previously described [44 (link)]. Briefly, total protein was isolated with RIPA Lysis Buffer (Upstate Biotechnology, New York, USA). The concentration of the protein was determined by protein assay reagents (Sigma-Aldrich, St. Louis, MO, USA) with a spectrophotometer (Beckman DU-640, Beckman Instruments, Inc., Indianapolis, IN). 20–30 μg of protein extracted from tumor tissues or cell lysates were separated by SDS-PAGE electrophoresis and then transferred onto nitrocellulose membranes. Membranes were then blocked with 5% non-fat milk in 1× PBS-T and then probed with a specific primary antibody to human EZH2 (1:1000 dilution, Cell Signaling Technology) or mouse monoclonal antibody to human β-actin (Sigma-Aldrich, St. Louis, MO, USA) at 4°C overnight. The membranes were further probed with respective secondary antibodies (LI-COR Biosciences, Lincoln, NE) and scanned by Odyssey® CLx equipment (LI-COR Biosciences) to detect the bands and the density. All other antibodies used in this study were purchased from Santa Cruz Biotechnology or Cell Signaling Technologies. Densitometric analysis of Western blot results was performed with Image J as described in the software's User Guide. The relative expression of protein was normalized to its respective actin expression.
3
Western Blot Analysis of ABC Transporters
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To evaluate the protein expression of Pgp and other ABC transporters including MRP1 and BCRP in different selected cell sublines, total proteins were extracted with 1 × RIPA lysis buffer (Upstate Biotechnology, Charlottesville, VA, USA). Protein concentrations were evaluated by the DC Protein Assay (Bio-Rad, Hercules, CA, USA), and western blot analysis was then performed as previously described (Duan et al, 2009b (link)). Briefly, 20 μg of total protein was resolved on NuPAGE 4–12% Bis-Tris Gel (Life Technologies) and transferred onto nitrocellulose membrane (Bio-Rad). The membrane was then blocked in Tris-buffered saline with 0.1% Tween-20 (TBST) containing 5% nonfat milk for 2 h and subsequently probed with primary antibodies (dilution: 1 : 1000) at 4 °C overnight. After washing three times with TBST, they were further incubated with respective secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA) in a dilution of 1 : 20 000 for 1 h at room temperature. The membranes were washed again with TBST and rinsed with PBS. Finally, membranes were scanned using Odyssey CLx equipment (LI-COR Biosciences).
4
Western Blot Analysis of Epigenetic Markers
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Protein lysates of the cells were extracted with 1× RIPA lysis buffer (Upstate Biotechnology, Charlottesville, VA) supplemented with complete protease inhibitor cocktail tablets (Roche Applied Science, IN, USA). The protein concentrations were determined by Protein Assay Reagents (Bio-Rad, Hercules, USA). Equal amounts of protein were separated by NuPAGE® 4–12% Bis-Tris Gel (Life Technologies), transferred onto nitrocellulose membrane (Bio-Rad), and incubated with specific primary antibodies EZH2 (Cell Signaling, Beverly, MA, dilution 1:1000), H3K27me3 (Active Motif, Carlsbad, CA, dilution 1:1000), and β-Actin (Sigma-Aldrich, St. Louis, MO, dilution 1:2000) at 4 °C overnight. The membranes were further probed with respective secondary antibodies (LI-COR Biosciences, Lincoln, NE), and scanned by Odyssey® CLx equipment (LI-COR Biosciences) to detect the bands and the density.
5
Silmitarsertib Cytotoxicity Assay
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IC50 of Silmitarsertib on SW480 wild-type and SW480 SALL2 KO cells were determined by crystal violet Assay. Five thousand cells were seeded on 96-well plates and incubated with increasing concentrations of Silmitarsertib (0–40 μM) for 24 h at 37 °C. Cells were stained with 0,01% (p/v) crystal violet [19 (link)] and fluorescence (680 nm) was quantified using LI-COR ODYSSEY CLX equipment.
6
Western Blot Analysis of Protein Extracts
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For western blot analysis, pellets containing ~2 × 106 test cells were resuspended in 20 μl of Laemmli 1 × buffer and incubated at 95°C for 10 min. Protein extracts were resolved by SDS-PAGE using 4/12% polyacrylamide gels under denaturing conditions. The resulting gel was washed with a 50% isopropanol solution containing 5% acetic acid for 15 min and subsequently washed with HPLC water for 15 min. Gels were blocked by incubation in 5% BSA solution containing 0.1% Tween 20 for 2 h. Primary labeling was performed by overnight incubation at 4°C with either Tau5 or anti-β-actin antibodies at a 1:2,000 dilution prepared in 5% BSA plus 0.1% Tween 20. Gel was washed three times with 1 × PBS plus 0.1% Tween 20, with a 10 min incubation in between. Secondary labeling was done with a CF™ 488A-labeled secondary antibody (Biotium, Fremont, CA, USA) at a 1:2,000 dilution, in which the gel was incubated for 2 h at room temperature and protected from the light. The gel was subsequently washed 3 times with 1 × PBS plus 0.1% Tween 20 for 10 min and once with 1 × PBS for 10 min. The gel was scanned in a LI-COR's Odyssey CLx equipment (Lincoln, NE, USA).
7
Western Blot Analysis of Protein Lysates
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Tissues and cells were lysed in RIPA buffer (Solarbio, China) with a cocktail inhibitor (Beyotime, China). Protein lysates were subjected to SDS-PAGE and transfer onto PVDF membranes (Millipore, USA) was then carried out. Membrane blocking conducted, followed by incubation overnight using primary antibodies at 4 °C. Antibodies used in this study were shown in Additional file 3: Table S2 . After being incubated using secondary antibodies, membranes were examined with Odyssey® CLx equipment (LI-COR, USA).
8
Enzalutamide Cytotoxicity in LOX-1 Knockdown Cells
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Enzalutamide (CAS No. 915087-33-1, Cayman Chemical) was dissolved at 40 mM in DMSO and stored at −20 °C. The cytotoxic effects of Enzalutamide on C4-2B and 22RV1 LOX-1 knockdown cells were determined by clonogenic assay. C4-2B and 22RV1 parental or LOX-1 knockdown cells were seeded on 6-well plates and pre-incubated with or without 50 µg/mL of oxLDL. Then, the cells were incubated with increasing concentrations of Enzalutamide (0–50 mM) and cultured for 14 days at 37 °C. Colonies were stained with crystal violet, images were photo-documented using the LI-COR ODYSSEY CLX equipment, and total colony numbers were counted for each condition.
9
Western Blot Analysis of Chordoma Proteins
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Cell pellets and human chordoma tissues were lysed in protein lysis buffer. The protein concentrations were calculated with Protein Assay Reagents (Bio-Rad, CA) and a SpectraMax 340PC Microplate Reader from Molecular Devices (San Jose, CA). Equal amounts of protein were separated on 4-12% Bis-Tris gels (NuPAGE®, Thermo Fisher Scientific, CA) and transferred to nitrocellulose membranes, where they were incubated with specific primary antibodies at 4°C overnight (CDK9 1:1000 dilution; RNAP II at 1: 1000 dilution; p-RNAP II Ser-2 at 1: 1000 dilution; Mcl-1 at 1: 1000 dilution; Bax at 1: 1000 dilution; Survivin at 1: 1000 dilution α -Tubulin at 1: 1000 dilution). After being washed with TBST three separate times for five minutes, the membranes were further incubated with Goat anti-rabbit IRDye 800CW or Goat anti-mouse IRDye 680LT secondary antibody (LI-COR Biosciences, NE. Final images were obtained with Odyssey® CLx equipment (LI-COR Biosciences). The abundance of α-Tubulin was monitored to ensure equal loading.
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