Exosomal protein concentration was determined by the Bradford method and proteins were separated by SDS-PAGE gel electrophoresis. Proteins were transferred onto PVDF membranes (Millipore, Burlington, MA, USA). The blots were blocked for 2 h at room temperature with 5% non-fat milk in PBS/0.05% Tween 20 and then incubated with the primary antibody (1 µg/mL) overnight at 4 °C. After washing, the PVDF membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h. The transferred proteins were visualized with chemiluminescence detection kit (Millipore, Burlington, MA, USA).
Chemiluminescence detection kit
Manufactured by Merck Group
Sourced in United States
The Chemiluminescence detection kit is a laboratory equipment product that enables the detection and quantification of chemiluminescent signals. It provides the necessary components and reagents to perform highly sensitive and specific chemiluminescence-based analyses.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Pvdf membrane, by Merck Group (4 mentions)
β actin, by Cell Signaling Technology (3 mentions)
G box imaging system, by Syngene (3 mentions)
Peroxydase conjugated secondary antibodies, by Jackson ImmunoResearch (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Cocktail of proteases and phosphatases inhibitors, by Merck Group (2 mentions)
Eb1 clone 5, by BD (2 mentions)
Ser 9 phospho gsk3β, by Cell Signaling Technology (2 mentions)
Gapdh, by Merck Group (2 mentions)
Protein assay, by Bio-Rad (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Anti mouse igg horseradish peroxidase, by Jackson ImmunoResearch (2 mentions)
Ibright fl1500 chemiluminescence imaging system, by Thermo Fisher Scientific (2 mentions)
α tubulin clone dm1a, by Merck Group (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Merck Group (2 mentions)
Total akt, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Bca kit, by Beyotime (2 mentions)
48 protocols using chemiluminescence detection kit
1
Exosomal Protein Quantification and Western Blot
2
PKCε Activation and Oxidative Stress
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Culture media were precipitated with trichloroacetic acid (1 : 100, vol/vol, overnight incubation at −20°C). The precipitates were rinsed with acetone, prior to be resuspended into lysis buffer. Proteins extracted from cells or heart tissues (40 μg protein for each sample) were electrophoresed on a SDS-PAGE and transferred onto a PVDF membranes (Bio-Rad) and incubated with primary antibodies against phosphorylated PKCε (1 : 500, Santa Cruz, CA, USA), PKCε, cleaved caspase-3, β-actin (1 : 1000, all from Cell Signaling Technology, Danvers, MA, USA), or 4-HNE (1 : 500) (both from Abcam, Cambridge, MA, USA). Then, membranes were incubated with HRP-conjugated secondary antibodies and exposed with the Chemiluminescence Detection Kit (Millipore).
3
Western Blot Analysis of Protein Expression
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Cells were seeded in a 6 cm dish (TPP, Trasadingen, Switzerland) and incubated with GRC-2 and prostratin at 37 °C and 5% CO2 in a cell culture incubator. Cell extracts were harvested in lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton-X100, 1 X protease inhibitor and 1 X phosphatase inhibitor) and the protein concentration was determined with protein assay dye reagent (Bio-Rad Laboratories, Richmond, CA, USA). Protein extracts were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 5% BSA for 1 h and incubated with specific primary antibody for overnight. Then, the membrane was incubated with HRP-conjugated secondary antibody for 1 h. Finally, the membrane was probed by the chemiluminescence detection kit (Millipore, MA, USA) and visualized with a LAS-4000 mini imaging system (Fujifilm, Tokyo, Japan).
4
Quantification of Protein Expression
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Cells were lysed after 6 h treatment in RIPA buffer (Tris-HCl 50 mM pH 8.0, NaCl 250 mM, Triton-X100 0.1%) with a cocktail of proteases and phosphatases inhibitors (Sigma-Aldrich) added freshly. Protein concentrations were determined using the Bio-Rad Protein Assay (Bio-Rad laboratories, Marnes-la-Coquette, France). Thirty micrograms of total proteins were loaded into a 12% SDS-PAGE gel and electrotransferred onto a nitrocellulose membrane. Primary antibodies used were directed against EB1 (clone 5; BD Biosciences, San Jose, CA), ATP1-A1 (Proteintech Europe, Manchester, UK), Ser 9 phospho-GSK3β (Cell Signaling, Boston, USA), total GSK3β (Thermo Fisher Scientific) and GAPDH (Sigma-Aldrich). Peroxydase-conjugated secondary antibodies (Jackson Immunoresearch, Baltimore, USA) and chemiluminescence detection kit (Millipore, Molsheim, France) was used for visualization of protein bands. Chemiluminescent signal was acquired on a G:BOX imaging system (Syngene, Cambridge, UK) and quantification was done with Image J software.
5
Western Blot Analysis of Apoptosis and Signaling
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Cells lysates were prepared with radioimmunoprecipitation assay buffer (RIPA) lysis buffer supplement with protease inhibitor cocktail (Calbiochem) and phosphatase inhibitor (Roche). The proteins are electrophoresed on 6–15% SDS-polyacrylamide gel (Beyotime Biotechnology) and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked in the TBS solution (100 mM Tris, 150 mM NaCl, pH 7.6) containing 5% (w/v) skim milk, and 0.5% (v/v) Tween-20 for 1 hour at room temperature. The membranes were incubated with primary antibodies overnight at 4°C followed by incubation with secondary antibodies for 1 hour. Primary antibodies used in this study were antibodies against caspase-3, -8, -9, Bax, pEGFR, pVEGF-R2, pAKT, AKT, and NK-κB, metal matrix protease (MMP) 2, MMP9, β-actin (Cell Signaling Technology, USA), pERK, and ERK (Abcam, UK). Goat polyclonal anti-mouse IgG-horseradish peroxidase (HRP) or goat polyclonal anti-rabbit IgG-HRP were used as secondary antibodies (Abcam, UK). Bands were visualized by chemiluminescence detection kit (Millipore, USA). All experiments were repeated 3 times.
6
Exosomal Protein Quantification and Immunoblotting
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Exosomal protein concentrations were quantified using a BCA protein assay kit (Pierce, FL, USA) following the manufacturer’s protocol. Equal amounts of protein (20 μg) were vortexed in 5 × loading buffer and denatured at 95 °C for 5 min, separated by 10% SDS PAGE, and transferred onto polyvinylidene fluoride (PVDF) membranes by electroblotting. The membranes were then blocked with 5% BSA for 1 h at room temperature and incubated overnight at 4 °C with mouse anti-CD63 (1:200 dilution; Santa Cruz, CA, USA), mouse anti-ALIX (1:200 dilution; Santa Cruz, CA, USA), mouse anti-TSG101 (1:200 dilution; Santa Cruz, CA, USA), or rabbit anti-calnexin (1:1000 dilution; Promega, Madison, WI, USA) antibodies. After incubation with a specific secondary anti-mouse or -rabbit horseradish peroxidase-conjugated antibody (1:5000 dilution; GeneTex, USA) at room temperature for 1 h, the protein bands were detected using a chemiluminescence detection kit (Millipore Co., MA, USA) and scanned using the iBright FL1500 chemiluminescence imaging system (Thermo Fisher Scientific, USA).
7
Western Blot Analysis of Akt Signaling
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Total cell protein extracts were separated by SDS/10% polyacrylamide gel electrophoresis, and transferred onto a polyvinylidene difluoride membrane (Millipore, U.S.A.). The membrane was blocked for 1 h in PBST with 5% non-fat milk at 4°C. Then, the blots were incubated with primary antibodies against Akt (SantaCruz), p-Akt (Ser473) (SantaCruz), PETN (SantaCruz), GAPDH (Cell Signaling) followed by horseradish peroxidase-conjugated secondary antibody and detected by chemiluminescence detection kit (Millipore, Billerico, Massochusatts, U.S.A.).
8
Protein Expression Analysis by Western Blot
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Total cell protein extracts were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto a polyvinylidene difluoride membrane. After blocking with 10% nonfat milk in phosphate-buffered saline, the membranes were immunoblotted with antibodies as indicated, followed by horseradish peroxidase-linked secondary antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA). The signals were detected using a chemiluminescence detection kit (Millipore, Billerica, MA, USA). Anti-dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX-1) and anti-GAPDH antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-PSA, CDK1 and CDK2 antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz Biotechnology, CA, USA). Protein levels were normalized against those of GAPDH (Santa Cruz Biotechnology, Inc.).
9
Immunofluorescence and Western Blotting of EB1 and Tubulin
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Indirect immunofluorescence was performed as previously described [5 (link)] by using the anti-EB1 antibody and anti-mouse antibody Alexa 568 nm (Molecular Probes); and FITC-coupled anti-α-tubulin antibody (clone DM1A; Sigma-Aldrich). Cells were observed using a Leica DM-IRBE microscope, 100X magnification. All images were acquired using Metamoph software (Molecular Devices, Sunnyvale, CA) at identical acquisition settings, and were processed using Image J software. After cell lysis, 30 μg of total protein were loaded into a 12% SDS-PAGE gel. Anti-EB1 antibody, anti-α-tubulin and anti-mouse IgG-horseradish peroxidase (Jackson Immunoresearch) were used. Chemiluminescence detection kit (Millipore) was used for visualization of protein bands. Chemiluminescent signal was acquired on a G:BOX imaging system (Syngene, Cambridge, UK) and quantification was done with Image J software.
10
Protein Expression Profiling in PC12 Cells
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Total proteins were obtained from PC12 cell lines by radioimmunoprecipitation assay buffer (RIPA) lysis buffer after being washed with cold phosphate-buffered saline (PBS). Total protein extract was fractionated with 10% SDS-PAGE and transferred onto PVDF membranes. Each membrane was incubated with specific primary antibodies overnight at 4°C after being blocked with 5% skim milk. Rabbit anti- NDRG4 antibodies (1: 1000), rabbit anti-p-eNOS antibodies (1: 1000), rabbit anti-eNOS antibodies (1: 1000), rabbit anti-iNOS antibodies (1: 1000), rabbit anti-Bcl2 antibodies (1: 1000), rabbit anti-Bax antibodies (1: 1000), rabbit anti-cleaved caspase-3 antibodies (1: 1000), and rabbit anti-caspase-3 antibodies (1: 1000) were acquired from Cell Signaling Technology (Danvers, MA, USA). After being washed with 1×TBST, 3 times, the PVDF membranes were reacted with appropriate horseradish peroxidase-conjugated secondary antibodies for 2 hours at room temperature. Further, the target proteins expression was analyzed by using chemiluminescence detection kit (Millipore, Billerica, MA, USA).
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