Cell lysates were prepared using RIPA lysis buffer (Pierce Technologies) and protease and phosphatase inhibitor (Thermo Scientific). Proteins were separated in 12% SDS-PAGE gel and transferred onto a nitrocellulose membrane and blocked with milk protein. Membranes were incubated with anti-Scx (1:1000, Rabbit polyclonal, Santa Cruz Biotechnology), anti-Tnmd (1:1000, Rabbit polyclonal, Santa Cruz Biotechnology), anti-phospho-Smad 1/5/8 (1:1000, Rabbit polyclonal, Millipore), anti-Smad8 (1:1000, Rabbit polyclonal, Santa Cruz Biotechnology), or α-Tubulin (1:1000, Rabbit polyclonal, Santa Cruz Biotechnology) followed by incubation with anti-rabbit conjugated with horseradish peroxidase (Santa Cruz Technologies). Membranes were washed with TBST and immunoreactivity was normalized by chemiluminescence using ECL Plus Detection system (Amersham Biosciences).
Ecl plus detection system
Manufactured by Cytiva
Sourced in United Kingdom, Switzerland, United States
The ECL Plus detection system is a chemiluminescence-based detection method used for the sensitive and quantitative analysis of proteins in Western blot experiments. The system utilizes a two-component detection reagent that produces a stable luminescent signal, allowing for the visualization and quantification of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Hybond, by Cytiva (5 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Murine monoclonal anti β actin, by Merck Group (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Cell lysis buffer, by Cell Signaling Technology (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Pvdf membrane, by Cytiva (2 mentions)
Hybond p pvdf, by Cytiva (2 mentions)
Hybond p, by Cytiva (2 mentions)
Complete mini, by Roche (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
α tubulin, by Santa Cruz Biotechnology (1 mentions)
Anti scx, by Santa Cruz Biotechnology (1 mentions)
Anti tnmd, by Santa Cruz Biotechnology (1 mentions)
Anti smad8, by Santa Cruz Biotechnology (1 mentions)
Anti phospho smad 1 5 8, by Merck Group (1 mentions)
Quantity one 4, by Bio-Rad (1 mentions)
38 protocols using ecl plus detection system
1
Western Blotting of Tendon-Related Proteins
2
Quantitative Western Blot Analysis
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Samples (30 μg) were run out in 10% SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and blocked in Tris-buffered saline (10 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 8.00) with 0.05% Tween 20 (TBS-T) containing 5% non-fat dry milk for 1 h at RT. Blots were then incubated overnight at 4 °C with rabbit polyclonal anti-human AQP11 (HPA042879, Sigma) or murine monoclonal anti-β-actin (Sigma) antibodies (diluted 1:1000 and 1:5000, respectively, in blocking solution). The antigen-antibody complexes were visualized using horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibodies (diluted 1:5000 in blocking solution) and the enhanced chemiluminescence ECL Plus detection system (Amersham Biosciences, Buckinghamshire, UK). The band intensities were determined by densitometric analysis with the Gel DocTM system and Quantity One 4.5.0 software (Bio-Rad) and normalized with β-actin density values.
3
Western Blot Analysis of PI3K/Akt Signaling
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Western blotting was performed as described previously [19 ]. Washed platelets (2–3X106 cells) in 1% IGEPAL NP40 and 100 mM protease inhibitor mix (Calbiochem; San Diego, CA, USA) were precipitated and separated under reducing and denaturalizing conditions by 10% polyacrylamide gel electrophoresis. The proteins were blotted to polyvinylidene (PVDF) membranes (BioRad; Hercules, CA, USA) and Western blot analysis was performed by immunoblotting with the following antibodies: PI3K (sites Tyr 458 and Tyr 199), P-PI3K, Akt (site Thr 308), P-Akt, or β-actin at 1:1000 dilutions (all antibodies were from Santa Cruz; CA, USA). The hybridization bands were revealed with the corresponding secondary antibodies (1:2000 dilutions) conjugated with horseradish peroxidase (Santa Cruz) and the ECL-plus detection system (Amersham, Buckinghamshire, UK). Densitometric analyses were performed using the Image J Software (NIH; Bethesda, MD, USA) and double normalization (100% intensity) was done by using α-tubulin as loading control, calculating the percentage of phosphorylated protein as phosphorylated protein/tubulin ratio. The percentage level of each enzyme or transporter shown in results represents the mean ± SD of at least three independent experiments.
4
Immunoprecipitation and Western Blot Analysis
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Cells lysates were subjected to immuno-precipitation (IP) and Western blot analysis as described 13 (link). Primary antibodies were bought from Cell Signaling Technology Inc. (Beverly, MA) (EGFR, IGF-1R, Akt, MAPK) and from Chemicon, Inc. (Pittsburgh, PA) (β-actin, α-tubulin). Protein A Sepharose beads were from GE Healthcare Biosciences (Uppsala, Sweden). Secondary antibodies were from Amersham Biosciences (Arlington Heights, IL). Immunoreactions were visualized using ECL-Plus detection system from Amersham Biosciences (Arlington Heights, IL) and analyzed by Typhoon scanner with ImageQuant software from Molecular Dynamics Inc. (Sunnyvale, CA).
5
Protein Extraction and Western Blotting
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Total proteins, cytosolic and nuclear extracts were prepared as previously reported [24] (link), and subjected to SDS-PAGE. Western blotting filters were developed using the ECL-plus detection system (Amersham, Dubendorf, Switzerland) or the SuperSignal West Femto kit (Pierce, Rockford, IL). The Abs employed for the study were as follows: anti-phospho-STAT3 (Serine 727, Ser727), anti-STAT3 (C-20), anti-phospho-STAT1 (Tyrosine 701, Tyr701), anti-STAT1 (E-23), anti-phospho-ERK1/2 (E-4), anti-ERK1/2 (C16), anti-phospho-EGFR (Tyr1173), anti-EGFR (1005), anti-Akt (H-136), anti-PCNA (PC10), anti-cyclin D1 (DCS-6), anti-β-actin (C-11), all provided by Santa Cruz Biotechnology (Santa Cruz, CA). anti-phospho-STAT3 (Tyr705), anti-acetyl-STAT3 (lysine 685, Lys685), anti-phospho-STAT1 (Ser727), anti-phospho-Akt (Ser473), and anti-phospho-Retinoblastoma (RB) (Ser795) were from Cell Signaling Technology (Denvers, MA). Filters were properly developed with anti-mouse or anti-rabbit Ig Abs conjugated to HRP.
6
Honokiol Modulates Protein Expression in Melanoma
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Western blotting was used to determine the levels of protein expression in SKMEL-2 and UACC-62 cells treated with varying concentrations of honokiol. SKMEL-2 or UACC-62 cells (1.5 × 106) were plated in 100 mm culture dishes. The cells were treated with honokiol 0 μM, 25 μM, 50 μM, 75 μM, or 100 μM for 12 and 24 hours. After each treatment, cells were lysed and protein concentrations were determined using BCA protein assay kit (Pierce, Rockford, FL, USA). Equal amounts of proteins were denatured and separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were blocked in 5% nonfat milk and incubated with the appropriate primary antibodies followed by secondary antibody. The proteins were detected using the ECL Plus Detection System (Amersham Biosciences, Piscataway, NJ). The band densities were quantified using the UVP Biochem Gel Documentation System (UVP Inc., Upland, CA, USA). Consistent protein loading was ensured by probing each membrane for β-actin. The Western blots were repeated 3–5 times. A representative blot is reported.
7
Western Blot Analysis of Phosphorylated ERK
8
Quantifying AKT Phosphorylation in Muscle Tissue
9
Immunoprecipitation and Immunoblotting Techniques
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Total protein extracts were prepared as previously reported [27 ]. Immunoprecipitation and immunoblotting were performed accordingly to standard procedures [27 ]. The Abs employed for the study were as follows: anti-IL-22R1, anti-phospho-STAT3 (Ser727), anti-STAT3 (C-20), anti-phospho-STAT1 (Tyr701), anti-STAT1 (E-23), anti-phospho-Erk1/2 (E-4), anti-Erk1/2 (C16), anti-β-actin (C-11), and HRP-conjugated anti-c-myc (9E10), all provided by Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-STAT3 (Tyr705) anti-MEK (mitogen-activated protein kinase) 1/2 and anti-phospho MEK1/2 (Ser218/222) were from Cell Signaling Technology (Denvers, MA); anti-SOCS1 and anti-SOCS3 were from MBL (Sigma-Aldrich, Milan, Italy), whereas anti-JAK1 and anti-phospho Tyr were from Upstate (Millipore, Milan, Italy). Filters were properly developed with anti-mouse, anti-goat, or anti-rabbit Ig Abs conjugated to HRP using the ECL-plus detection system (Amersham, Dubendorf, Switzerland), or, otherwise, the SuperSignal West Femto kit (Pierce, Rockford, IL, USA).
10
Western Blot Protocol for Oocyte Protein Analysis
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Sample preparation for western blotting was conducted as described44 (link). Briefly, denuded oocytes (n = 50) were collected and frozen in Laemmli buffer with protease inhibitors (Sigma). Prior to gel electrophoresis, samples were boiled at 100 °C for 5 min and then separated in 7.5% acrylamide gels containing 0.1% SDS. Proteins were transferred onto hydrophobic PVDF membranes (Amersham). Membranes were blocked with 5% non-fat milk in PBST (PBS with 0.1% Tween-20) overnight at 4 °C and then incubated with rabbit anti-LSH antibody (a gift from Dr. Kathrin Muegge, National Cancer Institute, 1/5000 in PBST) or anti-H3T3ph (EMD Millipore, 1:1000 dilution) at 4 °C overnight and subsequently washed in PBST. After three washes in PBST for a total of 60 min, the membranes were labeled with peroxidase-conjugated goat anti-rabbit IgG (Invitrogen, 1/5000 in PBST) for 1 h. The proteins were visualized using an ECL-plus detection system (Amersham). Some membranes were also probed with anti-β tubulin (Sigma Aldrich, 1/2000 dilution) as an internal control. Uncropped/unprocessed scans of all western blots can be found in the Source Data file.
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