Western blot analysis was performed as previously described [64 (link)]. Briefly, cells were lysed in cold lysis buffer, proteins (20–30 μg) were resolved on SDS-PAGE, transferred onto PVDF membranes, and probed with antibodies for FZD7 (sc-293261, Santa Cruz Biotechnology), TAZ (sc-48805, Santa Cruz Biotechnology), and GAPDH (sc-32233, Santa Cruz Biotechnology) at 4°C overnight. Detection was performed with the SuperSignal West Femto Maximum Sensitivity Substrate Trial Kit (Pierce, Rockford, IL, USA). The band images were digitally captured and quantified with a FluorChem FC2 imaging system (Alpha Innotech, San Leandro, CA, USA).
Fluorchem fc2
Manufactured by Bio-Techne
Sourced in United States
The FluorChem FC2 is a high-performance fluorescence and chemiluminescence imaging system designed for a wide range of life science applications. The system combines a sensitive CCD camera with advanced optics and software to capture and analyze images of fluorescent and chemiluminescent samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (6 mentions)
Polyvinylidene difluoride membrane, by Merck Group (4 mentions)
Anti β actin antibody, by Abcam (4 mentions)
Clarity western ecl substrate, by Bio-Rad (4 mentions)
P stat3, by Cell Signaling Technology (3 mentions)
Stat3, by Cell Signaling Technology (3 mentions)
Anti cd133, by Abcam (3 mentions)
Anti mouse secondary antibodies, by Abcam (3 mentions)
Anti cd44, by Abcam (3 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Stat1, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by GE Healthcare (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Horseradish peroxidase conjugated goat anti mouse igg, by Agilent Technologies (2 mentions)
Complete mini proteinase inhibitor cocktail tablets, by Roche (2 mentions)
Sc 48805, by Santa Cruz Biotechnology (1 mentions)
Sc 32233, by Santa Cruz Biotechnology (1 mentions)
Anti s100a4, by Abcam (1 mentions)
Anti ikk, by Abcam (1 mentions)
39 protocols using fluorchem fc2
1
Western Blot Analysis of FZD7, TAZ, and GAPDH
2
Protein Expression Analysis in Liver Tissues
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Liver tissues collected from different POD were treated with RIPA lysis buffer to extract total proteins, and the concentrations of the total proteins were detected using a BCA protein assay kit. The proteins were separated electrophoretically and then transferred to nitrocellulose membranes. After blocking with 5% skimmed milk for 2 h, ET-1 (1:250), eNOS (1:200), iNOS (1:250), vWF (1:250) and internal reference protein GAPDH (1:3000) antibodies were added and incubated at 4 °C overnight. The membranes were then rinsed with Tris Buffered Saline with Tween-20 (TBST), incubated with secondary antibodies (1:5000) for 2 h at room temperature, rinsed with TBST again, the chemiluminescence HRP substrate was added, and the membranes were exposed in a gel imaging analysis system (Alpha Innotech FluorChem FC2, CA, United States). The images were analyzed using the AlphaView SA 3.4.0 software (San Jose, CA, United States) to determine the grey scale. The relative abundance of a target protein was calculated as target protein band brightness value - background brightness value/internal reference protein GAPDH band brightness value - background brightness value. The resulting ratio was the relative abundance of the target protein. The samples were replicated three times in different batches at each time point.
3
Protein Expression in MB49 and MCSCs
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The MB49 cells and MCSCs were respectively harvested. Equal amount proteins were extracted from cells, and separated by 10% sodium dodecyl sulfate -polyacrylamide gel electrophoresis followed by transferring to polyvinylidene difluoride membranes (Millipore, Billerica, MA). The membranes were blocked with 5% skim milk in PBS, and incubated overnight at 4 °C with the primary antibodies including anti-S100A4 (Abcam, Cambridge, MA), anti- IKK (Abcam) and anti-β-actin antibody (Abcam) followed by secondary antibodies (Abcam). Bands were visualized using Fluor Chem FC2 (Alpha Innotech, San Leandro, CA).
4
Quantifying Ion Channel Expression
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The rCEnCs seeded with the density of 100 cells/mm2 films and TCP were cultured for 3 and 5 days. Trizol (Invitrogen, Carlsbad, CA, USA) and chloroform (Sigma-Aldrich, USA) were used to extract mRNA and centrifuged at 12,000 rpm in 4 °C for 15 min. The supernatant was transferred to a 1.5 mL Eppendorf tube. Iso-propanol (Sigma-Aldrich, USA) was added and kept in 4 °C overnight. Isolated mRNA was dissolved in RNase-DNase free water (Gibco, USA). The gene markers of voltage-dependent anion-selective channel 2 (VDAC2), voltage-dependent anion-selective channel 3 (VDAC3), chloride channel protein 2 (CLCN2), and sodium/bicarbonate co-transporter (NBC1) were evaluated and normalized using β-actin a housekeeping gene. Gene expression was measured by electrophoresis on 1% (w/v) agarose gel containing Ethidium Bromide (EtBr, Sigma-Aldrich, USA). Images were obtained under a UV light (FluorChem FC2, Alpha Innotech, San Leandro, USA) at 360 nm.
5
Western Blot Analysis of EMT Markers
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Cells were washed twice with ice-cold PBS and solubilized in RIPA buffer (Sigma-Aldrich, Saint Louis, MO) on ice and then was quantified using QuantiPro bicinchoninic acid assay kit (Sigma-Aldrich). Proteins were denatured at 100°C with sample buffer for 5 min. Equal amounts of protein (50 μg) separated by electrophoresis in 10% to 12% SDS-PAGE gels according to their molecular weight. Proteins were transferred onto PVDF membranes (Bio-Rad) and blocked for 2 h in blocking solution (5% nonfat dry milk in TBS containing 0.1% Tween 20). The membrane was then exposed to the primary antibody overnight at 4°C. Snail, slug, NF-κB, p-NFκB P65 (Ser536), STAT3, p-STAT3, AKT, p-AKT were purchased from Cell Signaling (Beverly, MA); Twist1, Zeb1, Zeb2, vimentin, E-cadherin, N-cadherin, β-action were purchased from Santa-Cruz Biotechnology, all primary antibodies dilution is 1:1000. After washing, the membranes were incubated for 1.5 h at room temperature with peroxidase-linked secondary antibody (Santa-Cruz). Signals were revealed with an electrochemoluminescence (ECL) Western Blotting Analysis System (Pierce) using the FluorChem®FC2 (Alpha Innotech, CA), and then quantified using ImageJ® software.
6
Western Blot Analysis of CD133 and CD44
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The protein extracts were separated by electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked and incubated using the primary antibody anti-CD133 (Abcam, Cambridge, MA, USA), anti-CD44 (Abcam) and anti-β-actin antibody (Abcam). Then membranes were incubated with anti-mouse secondary antibodies (Abcam). Finally, protein bands were detected using Fluor Chem FC2 (Alpha Innotech, San Leandro, CA, USA) and their intensity was analyzed using the Image Lab software.
7
Immunoblot Analysis of Palmitate-Treated Cells
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For immunoblot analysis, OR6 cells and cured cells were seeded on 10-cm plates and treated with 200–800 μM palmitate for 24 hours. Samples containing 50 μg protein were resolved by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and incubated with primary and then appropriate secondary antibodies. Bound antibodies were visualized with Clarity Western ECL Substrate (Bio RAD, Hercules, CA, USA) using a FluorChem® FC2 chemiluminescent imaging system (Alpha Innotech, San Leandro, CA, USA).
8
EphA2 mRNA Expression Quantification
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The total RNA of the tissue specimens was isolated by a SimplyP Total RNA Extraction kit (Bioer Technology Co. Ltd, Hangzhou, Zhejiang, China). The total RNA (1 μg) was reverse transcribed by High-Capacity cDNA Reverse Transcription kits (Applied Biosystems, Foster City, CA, USA) according to manufacturer’s instructions. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). The primers for EphA2 and GAPDH (internal control) were designed as follows: EphA2 forward, 5′-CCAAGTTCGCTGACATCGT-3′ and reverse, 5′-GCCATGAAGTGCTCCGTAT-3′; and GAPDH forward, 5′-ACCACAGTCCATGCCATCAC-3′ and reverse, 5′-TCC ACCACCCTGTTGCTGTA-3′. The RT product (1 μl) was amplified by PCR using the following conditions: One incubation of 5 min at 95°C for PCR; then 28 cycles for the EphA2 primers and 25 cycles for GAPDH at 95°C for 30 sec, 58°C for 45 sec and 72°C for 60 sec, and a final extension at 72°C for 7 min. The PCR product (8 μl) was then electrophoresed on 1.5% agarose gel, and the intensity of the bands was quantified by FluorChem FC2 (Alpha Innotech, San Leandro, CA, USA). EphA2 mRNA expression was determined as a relative intensity of the PCR product bands from the target sequences relative to the intensity of the GAPDH gene. PCR experiments were repeated three times.
9
Western Blot Analysis of Apoptotic Markers
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Protein extracts from the cells were prepared by washing the cells in PBS and incubating them in ice-cold lysis buffer supplemented with a protease inhibitor cocktail. After triple sonication and evaluation of the protein concentration, the protein samples (60 μg/lane) were subjected to 4–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to a nitrocellulose membrane for treatment with specific antibodies. Blots were developed using the alkaline phosphatase colorimetric system. The following primary antibodies were used: rabbit-anti Bim, rabbit-anti phospho-SAPK/JNK, rabbit-anti SAPK/JNK, rabbit-anti cleaved PARP, and rabbit-anti PARP. The antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Goat-anti actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Proteins were detected using horseradish peroxidase-conjugated rabbit anti-goat or goat anti-rabbit secondary antibodies (Life Technologies, Inc., Gaithersburg, MD, USA). The bound antibodies were visualized using a chemiluminescent substrate (SuperSignal® West Pico Chemiluminescent Substrate; Thermo Fisher Scientific, Rockford, IL, USA) and a chemiluminescent imaging system (FluorChem® FC2; Alpha Innotech, San Leandro, CA, USA).
10
Western Blot Analysis of Stem Cell Markers
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The protein extracts were separated by electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, MA, USA). Membranes were blocked and incubated using the primary antibody anti-CD133 (Abcam, MA, USA), anti-CD44 (Abcam) and anti-β-actin antibody (Abcam). Then membranes were incubated with anti-mouse secondary antibodies (Abcam) (7 (link)). Finally, protein bands were detected using Fluor Chem FC2 (Alpha Innotech, CA, USA) and their intensity was analyzed using the Image Lab software.
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