Balanced protein samples (30 μg per lane) were heated for 5 min at 95°C and then loaded on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, the gels were transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon-P, Millipore Corporation, Billerica, MA, USA) at 4°C by the wet transfer method (200 mA, 3 h). Following several rinses with phosphate buffer solution-Tween (PBST, containing 0.1% Tween-20), the transferred PVDF membrane was blocked with 5% nonfat milk in PBST for 1 h at room temperature. The membrane was then incubated for 3 h at room temperature with a rabbit polyclonal antibody against CRMP2 (Cell Signaling Technology, Danvers, MA, USA) and a mouse monoclonal antibody against αII-spectrin (Merck Millipore, Darmstadt, Germany). A mouse monoclonal antibody against β-actin (Santa Cruz Biotechnology, Paso Robles, CA, USA) was employed to verify equal loading of the samples. Horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Santa Cruz Biotechnology, Paso Robles, CA, USA) was used as the secondary antibody. After incubation with the primary and secondary antibodies, a chemiluminescent detection kit (Immobilon-P, Millipore Corporation, Billerica, MA, USA) was used to visualize the protein bands. The relative band intensities were tested by densitometry using FluorChem FC2 software (ProteinSimple, CA, USA).
Mouse monoclonal antibody against β actin
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Mouse monoclonal antibody against β-actin. This antibody recognizes the β-actin protein, a cytoskeletal protein expressed in eukaryotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Immobilon p, by Merck Group (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit or anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Oxamate, by Merck Group (1 mentions)
Mir 34a mimic, by GenePharma (1 mentions)
Mir 34a inhibitor, by GenePharma (1 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Ecl reagent, by Merck Group (1 mentions)
T7 endonuclease 1 t7e1, by New England Biolabs (1 mentions)
Phosphate buffered saline tablet, by Merck Group (1 mentions)
1 1 dioctadecyl 3 3 3 3 tetramethylindocarbocyanine perchlorate, by Merck Group (1 mentions)
Cholesterol, by Merck Group (1 mentions)
Soy phosphatidylcholine, by Avanti Polar Lipids (1 mentions)
Protein g agarose bead, by Thermo Fisher Scientific (1 mentions)
Bca reagent, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal antibody against flag, by Merck Group (1 mentions)
Anti flag antibody conjugated agarose beads, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
17 protocols using mouse monoclonal antibody against β actin
1
Western Blot Analysis of CRMP2 and α-Spectrin
2
Examining LDHA and Hexokinase 2 in miR-34a Modulation
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Rabbit monoclonal lactate dehydrogenase-A (LDHA) antibody was ordered from Cell Signaling (#2012); mouse monoclonal antibody against Hexokinase 2 was purchased from Santa Cruz (sc-6521); mouse monoclonal antibody against β-actin was purchased from Santa Cruz Biotechnology (sc-47778). Oxamate was purchased from Sigma–Aldrich (St. Louis, MO, U.S.A.). MiR-34a mimic, miR-34a inhibitor, and their corresponding negative control were purchased from Shanghai GenePharma Co. Ltd. (Shanghai, China).
3
Western Blot Analysis of Transgenic SCNT Calf Tissues
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Tissues (liver, lung, spleen, heart, ear skin, muscle, stomach, intestine, colon, kidney) from a transgenic SCNT calf and transfected bovine fetal fibroblasts expanded for 10 passages were extracted in lysis buffer (1% NP-40, 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 20 mM Tris–HCl, pH 7.4, 2 mM Na3VO4, 1 mM PMSF, and protease inhibitor cocktail). Proteins were quantified using a BCA Protein Assay kit. For western blot assays, equal amounts of protein (20 µg) were subjected to 12% SDS–PAGE and electrophoretically transferred to nitrocellulose (NC) membranes (0.45 µm, Millipore, Billerica, MA, USA). The non-specific sites on each blot were blocked with 3% BSA diluted in TBS with 0.05% Tween 20 (TBST) for 90 min. Proteins were detected using mouse monoclonal [9D3] antibody against IFNα-2b (Abcam, Cambridge, UK), purified mouse monoclonal antibody against LTF (Abgent, San Diego, CA, USA), or mouse monoclonal antibody against β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) as primary antibody and horseradish peroxidase (HRP)-conjugated goat anti-mouse Ig G (Jackson ImmunoResearch, West Grove, PA, USA) as secondary antibody. Proteins were detected using an enhanced chemiluminescence (ECL) reagent (Millipore).
4
SVV Infection in PK-15 Cells
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Porcine kidney PK-15 cells were cultured in DMEM/High Glucose supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. SVV was isolated previously and preserved by our lab [22 (link)]. T7 endonuclease 1 (T7E1) was purchased from New England Biolabs (NEB). Mouse polyclonal antibody against RIG-I was purchased from Cell Signaling Technology. Mouse monoclonal antibody against β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz). The rabbit polyclonal antibody against SVV viral structural protein VP2 was prepared by our lab.
5
Preparation and Characterization of Nanoparticles
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Rap (purity >99%) was kindly provided by Chong Kun Dang Pharm. Co. (Yongin, Korea). Soy phosphatidylcholine (SPC; purity >99%) and distearoylphosphatidylethanolamine-polyethylene glycol2000-folate (DSPE-PEG2000-Fol; DP2KF) were purchased from Avanti® Polar Lipids (Alabaster, AL, USA). 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), phosphate buffered saline (PBS) tablets, and cholesterol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Folic acid was purchased from Duksan Pure Chemical Co., Ltd. (Seoul, Korea). Acetonitrile, chloroform, dimethylsulfoxide, and other solvents purchased from commercial sources were of analytical or cell culture grade. The following antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): rabbit polyclonal antibodies against phosphorylated AMPKα (Thr172), phosphorylated mTOR (Ser2448), phosphorylated 4EBP-1, phosphorylated p70S6 (Ser371), phosphorylated ULK1 (Ser555 and Ser757), and cleaved PARP. Mouse monoclonal antibody against β-actin was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Rabbit polyclonal antibodies against LC3B, p62, and beclin-1 were purchased from Abcam (Cambridge, MA, USA).
6
Immunopurification and Proteomic Analysis
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Mouse monoclonal antibody against Flag, anti-Flag antibody conjugated agarose beads, dithiothreitol (DTT), iodoacetamide (IAA) were from Sigma-Aldrich (St. Louis, MO, USA). Mouse monoclonal antibody against β-actin was from Santa Cruz (Santa Cruz, CA, USA). Mouse monoclonal antibody against PKCζ and rabbit polyclonal antibody against PPP2CA were from Cell Signaling Technology (Danvers, MA, USA). Lipofectamine 2000, BCA reagents, and Protein G agarose beads were purchased from Invitrogen. Enhanced chemiluminescence reagents were from Pierce Biotechnology. Protease Inhibitor Cocktail tablets were from Roche Diagnostics (Indianapolis, IN, USA). Sequencing grade modified trypsin was purchased from Promega (Madison, WI, USA). LC-MS grade water and acetonitrile were bought from Merck (White-house Station, NJ, USA).
7
Evaluating EGFR-Akt-P38 Signaling Pathway
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High glucose medium DMEM, 0.25% trypsin, PBS, penicillin-streptomycin solution, and Giemsa solution were obtained from Gibco Company. Fetal calf serum (FCS) was purchased from Sijiqing Company, Hangzhou. DMSO and Annexin V-FITC /PI cell apoptosis kit were purchased from Kaiji Company, Nanjing. Mouse monoclonal antibody against p-EGFR, p-Akt, and p-P38, mouse monoclonal antibody against β-actin, and horseradish peroxidase (HRP)-conjugated anti-mouse antibody were purchased from Santa Cruz Company. C225 (2mg/ml) was provided by the German Merck Company, Flow cytometry (FACS Calibur, Beckman Coulter Company). Linear accelerate (ELEKTA Company).
8
Western Blot Analysis of JAM2 Protein
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Cells were homogenized in lysis buffer consisting of 50 mM Tris-HCl (pH 8.0), 150
mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, 1% sodium dodecyl sulfate
(SDS), and 25x protease inhibitor (Roche, Mannheim, Germany) for 30 min on ice.
Protein sample was mixed with 2× sample buffer (12.5 mM Tris-HCl (pH
6.8), 4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue) and
heated at 90°C for 5 min. Following separation by 10% SDS polyacrylamide
gel electrophoresis (SDS-PAGE), proteins on the gels were electrically
transferred to nitrocellulose membranes (Millipore, Bedford, MA). Then membranes
were incubated with the rabbit polyclonal anti-JAM2 antibody or mouse monoclonal
antibody against β-actin (1:800, Santa Cruz) in blocking buffer (4.9% BSA
in washing buffer) for overnight at 4°C. Then the membrane was incubated
in HRP-conjugated goat anti-mouse IgG for 1 h at 37°C. Membranes were the
visualized by ECL (BioFX, Aden Prairie, MN). Data were analyzed using ImageJ
program (NIH, Bethesda, MD).
mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, 1% sodium dodecyl sulfate
(SDS), and 25x protease inhibitor (Roche, Mannheim, Germany) for 30 min on ice.
Protein sample was mixed with 2× sample buffer (12.5 mM Tris-HCl (pH
6.8), 4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue) and
heated at 90°C for 5 min. Following separation by 10% SDS polyacrylamide
gel electrophoresis (SDS-PAGE), proteins on the gels were electrically
transferred to nitrocellulose membranes (Millipore, Bedford, MA). Then membranes
were incubated with the rabbit polyclonal anti-JAM2 antibody or mouse monoclonal
antibody against β-actin (1:800, Santa Cruz) in blocking buffer (4.9% BSA
in washing buffer) for overnight at 4°C. Then the membrane was incubated
in HRP-conjugated goat anti-mouse IgG for 1 h at 37°C. Membranes were the
visualized by ECL (BioFX, Aden Prairie, MN). Data were analyzed using ImageJ
program (NIH, Bethesda, MD).
9
EMMPRIN Regulation via ERK/IκB Pathway
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Fetal bovine serum (FBS) and Medium 199 (M-199) were purchased from Gibco BRL (Life Technologies, NY). Phorbol 12-myristate 13-acetate (PMA), H2O2, peptide-N-glycosidase F (PNGase F), swainsonine, calcein-AM and Coomassie blue were acquired from Sigma-Aldrich (St. Louis, MO); Endoglycosidase H (Endo H) from Hoffmann-La Roche (Basel Switzerland); TNF-α from eBioscience (San Diego, CA); PD98059 from Cell Signaling (Danvers, MA). For protein purification, the Immunopure rprotein A IgG plus orientation Kit and the silver staining kit were provided by Pierce (Rockford, IL); and the monoclonal antibody against EMMPRIN (mAb-EMMPRIN) was produced by Genscript Corporation (Nanjing, China). For Western blot, the protein extraction kit was acquired from Beyotime (Haimen, China); mouse monoclonal antibodies against EMMPRIN, pERK1/2, pIκBα, ERK1/2, IκBα and rabbit polyclonal antibodies against MMP9 from Abcam (Cambridge, MA); the mouse monoclonal antibody against β-actin from Santa Cruz (Dallas, Tex).
10
Osteogenic Differentiation Markers
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Sodium pyruvate, insulin, and fetal bovine serum (FBS) were purchased from Join Bio-Innovation (Seoul, Korea). Ascorbic acid and β-glycerophosphate were from Sigma (St. Louis, USA). Recombinant human BMP-7 (rhBMP-7) was purchased from R&D Systems (Minneapolis, MN, USA), and goat polyclonal antibodies against OPG, RANK, RANKL, TRAIL, and alkaline phosphatase (ALP), and mouse monoclonal antibody against β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
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