For the Western blotting, the following antibodies were used at the specified dilutions: α-c-Cbl (Cell Signaling, #2747, 1:1000), α-Fyn (Santa Cruz Biotechnology, SC-16, 1:1000), and α-Src (Santa Cruz Biotechnology, SC-19, 1:500), α-phospho-Tyr-416 (Cell Signaling, #2101, 1:1000), α-β-actin (Abcam, Ab 6276, 1:10,000). The α-Srcasm antibody used for Western blotting was a polyclonal antibody, generated by incubating rabbits with three purified glutathione S-transferase fusion proteins spanning murine Srcasm (aa 1–200, aa 150–400, and aa 389–474) was used as described previously.[15 (link)] The secondary antibody for all but β-actin was goat α-rabbit multi-HRP at 1:20,000 (Amersham). For β-actin, sheep α-mouse IgG-HRP (Amersham) was used at 1:10,000.
β actin
Manufactured by Cytiva
Sourced in United States
β-actin is a highly conserved cytoskeletal protein that is involved in cell motility, structure, and integrity. It is commonly used as a loading control and reference gene in various molecular biology techniques, such as Western blotting and quantitative PCR, to normalize target gene expression levels.
Automatically generated - may contain errors
Lab products found in correlation
Ecl detection system, by Cytiva (2 mentions)
β actin, by Merck Group (2 mentions)
Lysis buffer, by Abcam (2 mentions)
Bis tris polyacrylamide gel, by Thermo Fisher Scientific (2 mentions)
Lysis buffer, by Thermo Fisher Scientific (2 mentions)
Hif 1α and hydroxy hif 1α, by Abcam (2 mentions)
Enhanced chemiluminescence detection system, by Cytiva (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Horseradish peroxidase conjugated goat antirabbit igg or goat antimouse igg, by Cell Signaling Technology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Anti p p38, by ABclonal (1 mentions)
Bmp 2, by Bioss Antibodies (1 mentions)
Anti p38, by Proteintech (1 mentions)
Ecl system, by Cytiva (1 mentions)
Complete protease inhibitor cocktail tablet, by Roche (1 mentions)
Enhanced chemiluminescence western blotting kit, by Cytiva (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
V6630, by Merck Group (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
8 protocols using β actin
1
Western Blotting Antibody Dilutions
2
Exosome-derived Protein Analysis via Western Blot
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Proteins from exosome-rich fractions, myocardial tissues, or cells were extracted with Radio-Immunoprecipitation Assay (RIPA) buffer, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroblotting onto nitrocellulose membranes (Millipore). After being blocked with 5% skim milk for 1 hour, the membranes were incubated with primary antibodies against CD9, CD63, HSP70, CD81, Bcl-2, Bax, Cyclin D1, Wnt3a, Phospho-GSK-3β (Ser9) (p-GSK-3β), GSK-3β, β-catenin, and β-actin (Cell Signaling Technology, Danvers, MA) overnight at 4°C. Then the membranes were incubated with horseradish peroxidase–conjugated goat antirabbit IgG or goat antimouse IgG (Cell Signaling Technology) for 1 hour at room temperature. Proteins were visualized by the enhanced chemiluminescence detection system (Amersham, San Francisco, CA), and band intensities were quantified using ImageJ software from 3 independent results normalized by β-actin.
3
Quantitative Protein Analysis of BMP-2 and MAPK Signaling
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At each indicated time point, cells from each group were washed twice with ice-cold PBS supplemented with 1 mM sodium vanadate and lysed in modified radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1 mM EGTA, 50 mM Tris (pH 7.4), 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing a protease inhibitor cocktail (Complete Protease Inhibitor Cocktail Tablets; Roche Diagnostics Ltd., Taipei, Taiwan) and 1 mM sodium vanadate. The lysates were cleared by centrifugation at 14,000 rpm for 15 min at 4 °C. The proteins were quantitated using the BCA protein assay. The protein expression levels were analyzed by Western blotting using antibodies against BMP-2 (catalog number: bs-1012R; Bioss, Beijing, China), anti-p38 (catalog number: 14064-1-AP; Proteintech, Rosemont, IL, USA), anti-p-p38 (catalog number: AP0526; ABclonal, Wuhan, China), anti-JNK (catalog number: ARG51218; Arigo, Hsinchu, Taiwan), anti-p-JNK (catalog number: ARG51807; Arigo, Hsinchu, Taiwan) and β-actin (catalog number: A5441), and immunoreactions were visualized using an enhanced chemiluminescence (ECL) system (Amersham, UK).
4
Immunoblotting Analysis of Apoptosis Regulators
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The cell lysates were separated on 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (EMD Millipore Co., Hayward, CA, USA). The membranes were blocked with 5% skimmed milk for 1 h and incubated with rabbit monoclonal anti-Bcl-xL (cat. no. 2764), anti-Bcl-2-associated X protein (Bax) (cat. no. 5023) and anti-β-actin (cat. no. 4970) (Cell Signaling Technology, Inc., Danvers, MA, USA) antibodies at dilutions of 1:1,000 overnight at 4°C, respectively. Subsequently, the membranes were incubated with a polyclonal goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (dilution, 1:4,000; cat. no. 7074; Cell Signaling Technology, Inc.) for 1 h. Finally, enhanced chemiluminescence (Enhanced Chemiluminescence Western Blotting kit; Amersham Biosciences, Piscataway, NJ, USA) was used to visualize the results and β-actin was used as internal control.
5
Quantitative Protein Expression Analysis
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Western blot analysis was performed using antibodies against vimentin (1:1000, V6630, Sigma‐Aldrich), E‐cadherin (1:1000, Cell Signalling Technology, Danvers, MA), EGFR (1:100, Cell Signalling Technologies) and β‐actin (1:5000, Sigma‐Aldrich). The signals obtained were visualised with the enhanced chemiluminescence (ECL) detection system (Amersham) and normalised to β‐actin signal.
6
Western Blot Protein Analysis Protocol
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Crushed tissues were homogenized in lysis buffer (ThermoFisher) on ice for 1 min (20–30 strokes) using an automatic Dounce homogenizer set to 2000 RPM. Whole cell lysate was prepared in lysis buffer (Abcam) using 3 sets of 3-s sonication (power level 3) intervals on ice. Protein concentration was estimated using Pierce BCA assay kit and samples were prepared and heated for 90C for 5 min. Equal amounts of protein were loaded and separated on 4%–12% Bis-Tris polyacrylamide gel (Thermo Fisher Scientific), transferred to a PVDF membrane, blocked for 1–2 h using 5% fat free skim milk, washed and finally, probed with corresponding antibodies as specified below. Antibodies were from Cell Signaling Technology (HIF-1α and hydroxy HIF-1α dilution 1:3000, MCU dilution 1:5000, MICU1 dilution 1:3000), Abcam (CPT-1a, CPT-2, OXPHOS cocktail dilution 1:3000), ZYMED Laboratories (β-actin; dilution 1:1,000), and Amersham (secondary antibodies conjugated with peroxidase). Development was done using X-ray film using a series of timed exposures and ImageJ was used for densitometric analysis on scanned film images.74 (link)
7
Western Blot Protein Analysis Protocol
8
Western Blot Analysis of Lung Tissue
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Cells were lysed in cell lysis buffer, and lung tissues were homogenized in 0.25 m sucrose buffer containing protease inhibitor (Sigma, St. Louis, MO, USA P8340) and phosphatase inhibitor cocktails (Sigma, P5726) and centrifuged at 3000 g, 4°C, for 10 min and then in 100 000 g for 60 min. Westerns were conducted with supernatants as we have described previously (El‐Deiry et al., 1992 ; Disayabutr et al., 2016 ) with the following antibodies: PAI‐1 (Molecular Innovation, Novi, MI, USA ASMPAI‐GF, ASRPAI‐GF), α‐SMA (Biocare, CM001B), p53 (Santa Cruz, SC‐6243), p21 (Santa Cruz, Dallas, TX, USA SC‐397), procollagen 1α1 (Santa Cruz, SC‐8784‐R), procollagen 1α2 (Santa Cruz, SC‐8788), and β‐actin (Sigma, A5441). The protein bands were visualized using the ECL detection system (Amersham, Piscataway, NY, USA), semi‐quantified using ImageJ software, and normalized by β‐actin band intensity.
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