The following antibodies were used for western blot analysis: anti-NBPF15 rabbit polyclonal (1:500, 12848-1-AP, Proteintech, US), anti-NBPF7 goat monoclonal (1:500, sc-248056, Santa Cruz Biotechnology, US), anti-α-catenin mouse monoclonal (ab49105, Abcam, MA, US), anti-NF-κB (p65) mouse monoclonal (1:1000, Cat#6956, Cell Signaling Techology, MA, US), anti-IκBα mouse monoclonal (1:1000, Cat#4814, Cell Signaling Techology) and anti-β-actin mouse monoclonal (1:1000, Cat# A5441, Sigma-Aldrich, MO, US). Signals were developed with the enhanced chemiluminescence detection system (Pierce, Thermo Fischer Scientific, Bonn, Germany).
Mouse monoclonal anti β actin
Manufactured by Merck Group
Sourced in United States, United Kingdom, Canada, Germany
Mouse monoclonal anti-β-actin is a laboratory reagent used to detect and quantify the presence of the β-actin protein in biological samples. It is a highly specific antibody that binds to the β-actin protein, allowing for its identification and measurement in various assays.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (9 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (7 mentions)
Goat anti rabbit igg hrp, by Fortis Life Sciences (6 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Bovine serum albumin (bsa), by Merck Group (5 mentions)
Odyssey imaging system, by LI COR (5 mentions)
Goat anti mouse igg hrp, by Fortis Life Sciences (5 mentions)
Odyssey infrared imaging system, by LI COR (5 mentions)
Mouse monoclonal anti flag, by Merck Group (5 mentions)
Canada balsam, by Fujifilm (4 mentions)
Fluoroshield with dapi, by Merck Group (4 mentions)
Ecl prime reagent, by GE Healthcare (4 mentions)
Peroxidase conjugated secondary antibody, by Cell Signaling Technology (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Mouse monoclonal anti p53, by Santa Cruz Biotechnology (4 mentions)
Mouse monoclonal anti parp, by BD (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Chemidoc mp imaging system, by Bio-Rad (4 mentions)
Mouse monoclonal anti gapdh, by Santa Cruz Biotechnology (3 mentions)
242 protocols using mouse monoclonal anti β actin
1
Western Blot Analysis of Key Proteins
2
Western Blot Optimization and Quantification
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Proteins were size fractionated by SDS polyacrylamide gel electrophoresis followed by electroblotting to PVDF membranes. Proteins were visualized with the following antibodies: a rabbit peptide-specific antibody to IRF2BP2, described previously (11 (link)), anti-ELAVL1 mouse monoclonal (Abcam, #ab136542), anti-KHSRP mouse polyclonal (Abnova Corporation, #H00008570-A01), anti-AUF1 rabbit polyclonal (Abcam, #ab50692), anti-alpha-smooth muscle actin mouse monoclonal (Sigma, #A2547), anti-GAPDH mouse monoclonal (Santa Cruz Biotechnology, #sc-59540), and anti-β-actin mouse monoclonal (Sigma, #A2228). Primary antibodies were used at 1/5,000 dilution. The secondary horseradish peroxidase-conjugated antibodies used at a 1/10,000 dilution, goat anti-mouse IgG antibody (R&D Systems, #HAF007) and goat anti-rabbit IgG (H+L) antibody (Life Technologies, #31460), were revealed by chemiluminescence using the SuperSignal West Dura substrate (Thermo Fisher Scientific). Bands were quantified using the ImageJ software (33 (link)) with each lane signal normalized to loading control prior to fold conversion. Fold change compared to average of baseline values.
3
Immunocytochemical and Western Blot Analysis
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The primary antibodies used for immunocytochemistry were as follows: anti-Irgb10 rabbit polyclonal antiserum (64 (link)) at 1:1,000, anti-Gbp2 rabbit polyclonal (54 (link), 64 (link)) at 1:500, anti-LAMP1 mouse monoclonal (Santa Cruz Biotechnology) at 1:250, anti-LAMP1 rabbit polyclonal (Gbiosciences) at 1:250, anti-LAMP2 rabbit polyclonal (Invitrogen) at 1:250, and anti-LC3 rabbit polyclonal (MBL International) at 1:250. Protein samples from whole-cell lysates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The blots were probed with primary antibodies specific for the following: anti-Gbp2 rabbit polyclonal at 1:1,000, anti-Irgb10 rabbit polyclonal antiserum at 1:1,000, anti-hGBP1 rat monoclonal at 1:1,000 (Santa Cruz Biotechnology), and anti-β-actin mouse monoclonal (Sigma) at 1:5000. Binding of secondary horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG at 1:2,000 or goat anti-rat IgG at 1:2,000 or goat anti-mouse IgG antibodies at 1:5,000 (GeNei) was analyzed using Clarity Western ECL (Bio-Rad) or Westar Supernova chemiluminescent substrate (Cyanagen).
4
Western Blotting Protocol for Viral Protein Detection
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Cells were lysed in immunoprecipitation lysis buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol) containing protease inhibitors (Roche, Basel, Switzerland). Total cell lysates (25 μg) were boiled in SDS-PAGE loading buffer, subjected to SDS-PAGE, and subsequently transferred to a polyvinylidene fluoride membrane (Millipore-Sigma, St. Louis, MO, USA) using a semidry transfer apparatus (Bio-Rad, Hercules, CA, USA). The final dilution of the primary antibody was 1:5,000 for anti-K-Rta rabbit serum, 1 μg/ml anti-K8α (Santa Cruz, Santa Cruz, CA, USA), 1 μg/ml anti-ORF57 mouse monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA) and anti-LANA rat monoclonal (Millipore-Sigma, St. Louis, MO, USA), 1 μg/ml anti-K8.1 mouse monoclonal (Santa Cruz, Santa Cruz, CA, USA), 1:1,000 anti-RFP monoclonal antibody (Thermo Fisher), 1:500 anti-ORF52 monoclonal antibody, and 1:5,000 anti-β-actin mouse monoclonal (Millipore-Sigma, St. Louis, MO, USA). Washing membranes and secondary antibody incubations were performed as described previously (50 (link)).
5
Inflammasome Activation Protocol
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JT001 was synthesized at Jecure Therapeutics (San Diego, CA, USA). Emricasan was purchased from Selleckchem (Houston, TX, USA) and TAK-242 was purchased from Sigma-Aldrich (St. Louis, MO. USA). Nigericin, lipopolysaccharide (LPS) E. Coli O26:B6, Poly(dA:dT) and ATP were purchased from Sigma-Aldrich (St. Louis, MO, USA). Flagellin (FLA-BS Ultrapure), MSU, L18-MDP muramyl-dipeptide, and PAM3CSK4 were purchased from Invivogen (San Diego, CA, USA). FuGENE HD and CellTiter-Glo luminescent cell viability assay were purchased from Promega (Madison, WI, USA). The following primary antibodies were used: anti-ASC mouse monoclonal antibody (clone 2EI-7, Millipore-Sigma, St.
Louis, MO, USA), anti-MPO rabbit polyclonal antibody (ThermoFisher, Waltham, MA, USA), anti-procaspase-1 + p10 + p12 rabbit monoclonal (clone EPR16883, Abcam, Cambridge, UK), anti-αSMA rabbit polyclonal antibody (clone EPR5368, Abcam, Cambridge, UK), anti-NLRP3 mouse monoclonal (clone Cryo-2, Adipogen, San Diego, CA, USA) and anti-β-actin mouse monoclonal (clone AC-74, Millipore-Sigma, St. Louis, MO, USA). Secondary antibodies included goat anti-mouse IgG Alexa Fluor-488 (Life Technologies, Carlsbad, CA, USA) and horse-radish peroxidase-conjugated secondary antibodies (Azure Biosystems, Dublin, CA, USA).
Louis, MO, USA), anti-MPO rabbit polyclonal antibody (ThermoFisher, Waltham, MA, USA), anti-procaspase-1 + p10 + p12 rabbit monoclonal (clone EPR16883, Abcam, Cambridge, UK), anti-αSMA rabbit polyclonal antibody (clone EPR5368, Abcam, Cambridge, UK), anti-NLRP3 mouse monoclonal (clone Cryo-2, Adipogen, San Diego, CA, USA) and anti-β-actin mouse monoclonal (clone AC-74, Millipore-Sigma, St. Louis, MO, USA). Secondary antibodies included goat anti-mouse IgG Alexa Fluor-488 (Life Technologies, Carlsbad, CA, USA) and horse-radish peroxidase-conjugated secondary antibodies (Azure Biosystems, Dublin, CA, USA).
6
Western Blot Analysis of BBF2H7 and IL-6
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Proteins were extracted from cells using cell lysis buffer (1% Triton X-100, 100 mM EDTA, 50 mM NaCl, 10 mM HEPES, 500 mM sucrose, and a protease inhibitor cocktail [MBL International]). The protein concentrations of the cell lysates were determined using a Pierce BCA Protein Assay kit (Thermo). Equal amounts of protein were electrophoresed on 12% SDS-polyacrylamide gels, and then transferred onto polyvinylidene difluoride membranes (Bio-Rad). For immunoblotting, the following antibodies and dilutions were used: polyclonal rabbit anti-BBF2H7 C-terminus (Abcam; 1:1,000), polyclonal mouse anti-BBF2H7 N-terminus (the generated antibody [15 (link)]; 1:1,000), monoclonal mouse anti-β-actin (Sigma; 1:3,000), polyclonal rabbit anti-interleukin 6 (IL-6) (Abcam; 1:250), alkaline phosphatase-conjugated anti-rabbit IgG (Sigma; 1:3,000), and alkaline phosphatase-conjugated anti-mouse IgG (Enzo; 1:3,000). Labeled proteins were detected using nitro-blue tetrazolium chloride (WAKO) and 5-bromo-4-chloro-3'-indolylphosphatase p-toluidine salt (Roche).
7
Immunoblotting for Protein Expression
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Immunoblotting with primary antibodies (rabbit polyclonal anti-human IDO1, 1:1000, Cell Signaling Technology, Danvers, MA; monoclonal rat anti-mouse IDO1, 1:500, BioLegend, San Diego, CA; monoclonal rabbit anti-β-actin, 1:2000, Cell Signaling Technology; monoclonal mouse anti-β-actin, 1:4000, Sigma-Aldrich; rabbit monoclonal anti-vinculin, 1:1000, Cell Signaling Technology) was detected by chemiluminescence as previously described [36 (link)]. Alternatively, fluorescence was quantified on the Odyssey Imaging System (Li-Cor BioSciences, Lincoln, Nebraska).
8
Protein Expression Analysis by Western Blot
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Proteins were solubilized in Laemmli sample buffer, fractionated on SDS–PAGE (50 μg/well), and transferred to a nitrocellulose membrane (Thermo Scientific). Immunoblots were incubated overnight at 4°C with polyclonal rabbit anti‐OPN (1:2,000, Abcam), polyclonal goat anti-TNAP (1:2,000, R&D Systems) and monoclonal mouse anti-β-actin (1:5,000, Sigma) in 2% (w/v) bovine serum albumin (BSA, Sigma) in a TBS‐T buffer [150 mM NaCl, 10 mM Tris (pH 7.4/HCl), 0.2% (v/v) Tween-20]. After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Sigma). Specific signal was visualized by ECL kit (Amersham Life Sciences). The protein bands were quantified by Image Quant 5.0 software (Molecular Dynamics).
9
Gut Microbiome Modulation and Immune Response
10
Immunohistochemistry and Western Blotting Protocol
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