On the 3rd, 7th, and 14th days after treatment, granulation tissue under the new epithelium was taken from the infected wounds of the mice under sterile conditions. Nuclear and cytoplasmic protein lysates were prepared. The protein concentrations were measured with a BCA Protein Assay Kit (Beyotime, Nanjing, China). Equal amounts of protein (30 μg) were loaded into each lane of a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and separated. After the proteins had been transferred onto polyvinylidene difluoride membranes, the membranes were blocked and incubated with primary antibodies at 4°C overnight. The following primary antibodies were used: anti-TNF-α, anti-IL-1β, anti-TGFβ-1, and anti-VEGF antibodies from Abcam (United States) and anti-β-actin antibody from Bioworld (United States). After incubation with secondary antibody (goat anti-rabbit), the membranes were treated with an enhanced chemiluminescence reagent mixture (Thermo Fisher Scientific, United States) for 5 min and imaged on a Vilber Lourmat Fusion FX5 system (China).
Anti β actin antibody
Manufactured by Bioworld Technology
Sourced in United States
The Anti-β-actin antibody is a reagent used in various laboratory techniques for the detection and quantification of the beta-actin protein. Beta-actin is a ubiquitously expressed cytoskeletal protein that is commonly used as a loading control in Western blot and other protein analysis experiments.
Automatically generated - may contain errors
Lab products found in correlation
Anti pkm2 4053s, by Cell Signaling Technology (3 mentions)
Anti kif2c antibody, by Santa Cruz Biotechnology (3 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Bcl 3 polyclonal antibody, by Bioworld Technology (2 mentions)
Rrs1 polyclonal antibody, by Bioworld Technology (2 mentions)
Anti flag 8146s, by Cell Signaling Technology (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti il 1β, by Abcam (1 mentions)
Anti tnf α, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Anti tgf β1, by Abcam (1 mentions)
Normal mouse igg, by Merck Group (1 mentions)
Anti vimentin antibody, by Santa Cruz Biotechnology (1 mentions)
Anti bax, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Cyclin b, by Cell Signaling Technology (1 mentions)
Ecl plus western blotting detection reagents, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein quantification kit, by Vazyme (1 mentions)
12 protocols using anti β actin antibody
1
Inflammatory Cytokine Analysis in Wound Healing
2
Antibody-Mediated Interactions in EV-A71
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Anti-EV-A71 VP1 antibody was obtained from Abnova (Taiwan, China); anti-EV-A71 3c antibody, Abconal (Wuhan, China); anti-vimentin antibody, Santa Cruz (Dallas, Texas, USA); anti-β-actin antibody, anti-mouse and anti-rabbit secondary antibodies and electrochemiluminescent (ECL) Horseradish Peroxidase (HRP) substrate was used for western blotting, (Bioworld, Minneapolis, MN, USA); normal mouse IgG, Sigma (St. Louis, MO, USA); the Co-Immunoprecipitation (Co-IP) Kit, (Thermo Fisher, Rockford, IL, USA); the VIM (1–58) peptide, Ontores Biotech (Hangzhou, Zhejiang, China).
3
Western Blot and ELISA Analysis of Cell Signaling
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Cells with various treatments were lysed in an ice-cold RIPA lysis buffer (Beyotime), centrifuged to collect supernatants, and quantitated by BCA Protein Quantification Kit (Vazyme Biotech Co., Ltd.). Equal amounts of proteins were then fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to PVDF membrane (Bio-Rad, Hercules, CA), blocked in 5% BSA at room temperature for 1 h, and immunostained with primary antibodies (anti-Bax, Bcl-2, Caspase 3, Cyclin A, Cyclin B and CDK1 antibodies, Cell Signaling Technologies; anti-β-actin antibody, Bioworld; anti-MK antibody, Abcam) at 4 °C overnight and secondary antibodies (Thermofisher Scientific) at room temperature for 1 h. The expression of proteins was then visualized with ECL Plus western blotting detection reagents (Millipore, USA).
ELISA assay for MK in cell culture media MK in cell culture supernatant was measured via an enzyme-linked immunosorbent assay (ELISA) using a commercial kit (Abcam) according to the manufacturer's instructions. Briefly, after SKOV3 cells were treated with DHA, Cur, or both for 48 h, cell culture media were collected and centrifuged at 2, 000 × g for 10 min to remove debris. Aliquots of 50 µl diluted samples were then measured according to the manufacturer's instructions, and the final data were obtained from the detected OD at 450 nm.
ELISA assay for MK in cell culture media MK in cell culture supernatant was measured via an enzyme-linked immunosorbent assay (ELISA) using a commercial kit (Abcam) according to the manufacturer's instructions. Briefly, after SKOV3 cells were treated with DHA, Cur, or both for 48 h, cell culture media were collected and centrifuged at 2, 000 × g for 10 min to remove debris. Aliquots of 50 µl diluted samples were then measured according to the manufacturer's instructions, and the final data were obtained from the detected OD at 450 nm.
4
Protein Extraction and Western Blot Analysis
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Cells were lysed with modified RIPA buffer (50 mM of Tris, 150 mM of NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS) containing 25 µg/ml of leupeptin, 1 mM of sodium orthovanadate, 2 mM of EDTA and 1 mM of phenylmethylsulfonyl fluoride. The concentration of the protein samples was determined using a BCA kit (Beyotime Institute of Biotechnology, Beijing, China). A total of 20 µg of each protein sample was loaded onto an 8% SDS-PAGE gel, electrophoresed and blotted onto a polyvinylidene difluoride membrane (Sigma-Aldrich; Merck KGaA). The membranes were blotted with the first antibody (anti-p-p38) overnight at 4°C (cat. no. AM063; Beyotime, Zhejiang, China), anti-IDO (cat. no. H00003620-B01P; Abnova, Walnut, CA, USA), anti-Ku80 (cat. no. BS2692; Bioworld Technology, Inc., St. Louis Park, MN, USA) and anti-β-actin antibody (cat. no. MAB12983; Abnova) and with a secondary antibody at room temperature for 1 h. Immunoreactivity was detected using an enhanced chemiluminescence system (Xi'an Jiaotong University) and normalized to β-actin.
5
Protein Expression Analysis by Western Blot
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Cells were treated with pharmacological agents for various times. The cells were collected into lysis buffer. Equal amounts of total proteins (20 µg) were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were probed with the appropriate antibodies. The immunoreactivity was detected by ECL and analyzed using Image Lab software. The following were commercially obtained antibodies: the anti-PKM2 (#4053s), the anti-p62 (#8025), anti-LC3 (#12741) and anti-Flag (#8146s) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA); the anti-KIF2C antibody (#sc-81305) were obtained from Santa Cruz Biotechnology; the anti-β-actin antibody was obtained from Bioworld Technology (Atlanta, Georgia, 305, USA).
6
Therapeutic Compound Synthesis and Characterization
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Testosterone propionate, estradiol benzoate and olive oil were purchased from Aladdin Co., Ltd. (Shanghai, China). NAF (>99%) and HJZ-12 (>95%) were synthesized in according with the previous methods (Huang et al., 2015 (link)). Pierce BCA Protein Assay Kit was purchased from Thermo Fisher Scientific (Waltham, MA, United States). Anti-α-smooth muscle actin (α-SMA, 1/400) was purchased from Sigma-Aldrich (MO, United States). Anti-β-actin antibody (1/5000), Bcl-3 polyclonal antibody (1/500) and RRS1 polyclonal antibody (1/500) were purchased from Bioworld Technology (Inc., United States). Rabbit monoclonal anti-proliferating cell nuclear antigen (PCNA, 1/1000), and anti-FGFR3 antibody (1/1000) were purchased from Abcam (Cambridge, MA, United States). Anti-Bmi-1 antibody (1/1000) and anti-cleaved caspase 3 (1/1000) were purchased from Cell Signaling Technology (Beverly, MA, United States). All reagents were prepared fresh before use.
7
Western Blot Analysis of CIRP Protein
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Cells and tissues were lysed in a triple detergent RIPA buffer (Beyotime Institute of Biotechnology, Haimen, China) containing a protease inhibitor cocktail. Protein (~40 µg, which was determined by BCA method) was separated by 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% skimmed milk at room temperature for 1 h, prior to being incubated with an anti-CIRP antibody (cat. no., 10209-2-AP; dilution, 1:1,000; ProteinTech Group, Inc., Chicago, IL, USA) and either an anti-GAPDH antibody (for the cells; dilution; 1:3,000; Bioworld Technology, Inc., St. Louis Park, MN, USA) or an anti-β-actin antibody (for the tissues; dilution; 1:100; cat. no. ab8226; Abcam, Cambridge, UK) at 4°C overnight. Following incubation with the secondary antibody (fluorescein-conjugated goat anti-mouse IgG H&L; cat ab6785; Abcam) at room temperature for 45 min, signals were visualized using enhanced chemiluminescence [cat.no. NEL103001EA; Western Lightning® Plus-ECL, Enhanced Chemiluminescence Substrate, PerkinElmer, Inc., Waltham, MA, USA].
8
Western Blot Analysis of Protein Expression
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Cells were treated with pharmacological agents at various times, and subsequently collected into lysis buffer. MG132 and NS-398 were obtained from MedChemExpress (Shanghai, China). Equal amounts of total proteins (20 μg) were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and immunoblotted with appropriate antibodies. The immunoreactivity was detected by ECL and analyzed using Image J software. The following antibodies were commercially obtained: the anti-Nrf2 (#sc-365949) was obtained from Santa Cruz Biotechnology; the Anti-CUL3 (#10450) was obtained from Cell Signaling Technology (Danvers, MA, USA); the anti-α-SMA (#ab7817) was obtained from Abcam plc (Cambridge, CB2 0AX, UK). Anti-COX-2 (#160112) was from Cayman Chemical Co (Ann Arbor, MI, USA). Anti-KEAP1 (#A00514-3) and anti-FN1 (#BA1771) antibodies were obtained from Boster Biological Technology co. (Wuhan, China); anti-FAP-1 antibody (#SAB4500839) was obtained from Sigma Chemical Co. (St. Louis, MO, USA); the anti-β-actin antibody was obtained from Bioworld Technology (Atlanta, Georgia, 305, USA). Anti-CD9 (20597–1-AP), CD81 (66866–1-Ig), CD63 (67605–1-Ig), TGS101 (28283–1-AP) were obtained from Proteintech Group, Inc (Wuhan, China).
9
Wnt Signaling Pathway Analysis Protocol
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PD168368 was purchased from Tocris Bioscience. The antibodies against NMB and NMB receptor were supplied by Thermo Fisher Scientific and Santa Cruz Biotechnology, respectively. Antibodies against phospho-β-catenin (Ser33/37/Thr41), phospho-β-catenin (Ser675), β-catenin, phospho-GSK-3β (Ser9), GSK-3β, Runx2, total/cleaved-caspase-3, Bcl2, and Bad were procured from Cell Signaling Technology. Wnt3a and calponin antibodies were purchased from Abcam and anti β-actin antibody was from Bioworld Technology.
10
Quantitative Protein Analysis by Western Blot
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The total protein of concentration of each tissue or cell sample was determined by the Pierce BCA Protein Assay Kit (Waltham, MA, United States). 12% SDS-polyacrylamide gel and polyvinylidene-difluoride (PVDF) membranes (Millipore, Burlington, MA, United States) was used, and the membrane was blocked for 1 h at room temperature with 5% non-fat milk. The primary antibodies was incubated overnight at 4°C, including rabbit monoclonal anti-PCNA, anti-FGFR3 antibody; anti-β-actin antibody (1/5000), Bcl-3 polyclonal antibody and RRS1 polyclonal antibody (1/500; Bioworld Technology, Inc., United States); and anti-Bmi-1 antibody and anti-cleaved caspase3 (1/1000; CST, Beverly, MA). Then, the HRP-conjugated anti-mouse or anti-rabbit IgG (1/5000; Sigma–Aldrich MO, United States) was incubated for 1 h at room temperature. The enhanced chemiluminescence (ECL) western blotting detection reagents (GE Healthcare Biosciences, Pittsburgh, PA, United States) was used to get the band. The band was analyzed by ImageJ software.
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