Western blotting analysis was essentially carried out as described in our previous study [12 (link)]. Rabbit polyclonal antibody against GSE1 (1 : 1000, Proteintech Group, Inc., Chicago, USA) and mouse monoclonal antibody against β-actin (1 : 5000, Sigma) were used.
Mouse monoclonal antibody against β actin
Manufactured by Merck Group
Sourced in United States, United Kingdom, Italy
The mouse monoclonal antibody against β-actin is a laboratory reagent used for the detection and quantification of the β-actin protein in various biological samples. β-actin is a ubiquitous and highly conserved cytoskeletal protein found in all eukaryotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti srebp 1 antibody, by Santa Cruz Biotechnology (2 mentions)
Dual luciferase reporter assay kit, by Promega (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Sds page gel, by Bio-Rad (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Rabbit polyclonal antibody against gse1, by Proteintech (1 mentions)
Alexa 488 conjugated anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated secondary antibody, by Bio-Rad (1 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Ab5103, by Abcam (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Tris tricine ready gel, by Bio-Rad (1 mentions)
Enhanced chemiluminescence detection reagent, by Beyotime (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Image lab 3, by Bio-Rad (1 mentions)
P0012, by Beyotime (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal antibody against α smooth muscle actin α sma, by Merck Group (1 mentions)
Syto 61, by Thermo Fisher Scientific (1 mentions)
35 protocols using mouse monoclonal antibody against β actin
1
Western Blotting Analysis of GSE1
2
Immunochemical Analysis of Stress Response Proteins
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Antibodies used were: rabbit polyclonal antibody against GRP78 (AnaSpec Inc. Fremont, CA USA); goat polyclonal antibody against GRP78 (Santa Cruz Biotechnology, Inc. Dallas, TX, USA); rabbit polyclonal antibody against NAD(P)H:quinone oxidoreducrase1(NQO1) (Santa Cruz Biotechnology Inc.); mouse monoclonal antibody against NAD(P)H:quinone oxidoreducrase1(NQO1) (Novus Biologicals, LLC. Littleton, CO. USA); mouse monoclonal antibody against β-actin (Sigma-Aldrich Inc. St. Louis, MO, USA); Alexa 488-conjugated anti-rabbit antibody; Alexa 488-conjugated anti-mouse antibody (Invitrogen/Life Technologies); PE-conjugated anti-mouse secondary antibody (BioLegend Inc. San Diego, CA, USA); horseradish peroxidase (HRP)-conjugated goat polyclonal antibody against rabbit IgG; HRP-conjugated goat polyclonal antibody against mouse IgG; HRP-conjugated rabbit polyclonal antibody against goat IgG (DAKO Glostrup, Denmark).
3
Protein Expression Analysis of NOD1 Signaling
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After stimulation of primary cultures, cells were washed twice with ice-cold PBS. Whole protein extracts were obtained using ice-cold proprietary detergent in 25 mM bicine, 150 mM NaCl; pH 7.6 (T-PER® Tissue Protein Extraction Reagent, Thermo Fisher Sci.) supplemented with phosphatase cocktail and protease inhibitors (Sigma) [5 (link),18 (link)].
Proteins were resolved on SDS-PAGE gels and then transferred to nitrocellulose membranes. Proteins were detected using rabbit polyclonal antibody against NOD1 (1:500, Abcam), rabbit polyclonal antibody against phospho-RIP2 (1:1000, Cell Signaling), rabbit polyclonal antibody against RIP2 (1:1000, Cell Signaling), rabbit polyclonal antibody against phospho-NF-κB p65 (1:3000, Santa Cruz), mouse monoclonal antibody against NF-κB p65 (1:1000, Santa Cruz), mouse monoclonal antibody against β-actin (1: 40,000, Sigma), and HRP-conjugated secondary antibodies (BioRad, Hercules, CA, USA).
Protein bands were visualized using a Luminata chemiluminescence detection system (Merck Millipore, Madrid, Spain) and a ImageQuant LAS 500 imager (GE Healthcare Life Sci., Madrid, Spain) and were quantified using ImageJ (National Institutes of Health). Protein band intensities of interest were expressed as a percentage of those of the β-actin bands as indicated.
Proteins were resolved on SDS-PAGE gels and then transferred to nitrocellulose membranes. Proteins were detected using rabbit polyclonal antibody against NOD1 (1:500, Abcam), rabbit polyclonal antibody against phospho-RIP2 (1:1000, Cell Signaling), rabbit polyclonal antibody against RIP2 (1:1000, Cell Signaling), rabbit polyclonal antibody against phospho-NF-κB p65 (1:3000, Santa Cruz), mouse monoclonal antibody against NF-κB p65 (1:1000, Santa Cruz), mouse monoclonal antibody against β-actin (1: 40,000, Sigma), and HRP-conjugated secondary antibodies (BioRad, Hercules, CA, USA).
Protein bands were visualized using a Luminata chemiluminescence detection system (Merck Millipore, Madrid, Spain) and a ImageQuant LAS 500 imager (GE Healthcare Life Sci., Madrid, Spain) and were quantified using ImageJ (National Institutes of Health). Protein band intensities of interest were expressed as a percentage of those of the β-actin bands as indicated.
4
Western Blot Analysis of Histone H3 Citrullination
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Mice were terminally anesthetized and the lungs collected. Samples were homogenized in a lysis buffer containing a mixture of proteinase inhibitors (Tris-HCl 50 mM, pH 7.4; NP-40 1%; Na-deoxycholate 0.25%; NaCl 150 mM; EDTA 1 mM; PMSF 1 mM; aprotinin, leupeptin, and pepstatin 1 μg/ml). Proteins were separated by SDS-polyacrylamide gel electrophoresis and trans-blotted onto nitrocellulose membranes (Amersham Pharmacia Biotech). The membranes were blocked with 10% dry milk and incubated overnight at 4 °C with rabbit polyclonal antibody against Histone H3 citrulline R17+R2+R8 (1:200; ab5103, Abcam). The membranes were incubated with secondary antibody (Jackson ImmunoResearch). Immunodetection was performed using an enhanced chemiluminescence light-detecting kit (Amersham Pharmacia, Biotech) for 1 min. The mouse monoclonal antibody against β-actin (1:5000; Sigma Aldrich) was used for loading controls. Optical densitometry was measured following normalization to the control (house-keeping gene) using Scientific Imaging Systems (Image labTM 3.0 software, Biorad Laboratories, Hercules, CA, USA).
5
Western Blot Analysis of TIPE Protein
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All cells were harvested at approximately 80% confluence, and the protein was extracted. The protein concentration was determined using the BCA Protein Assay Kit (Pierce, Thermo Fisher Scientific). The protein (30 μg) samples were run on 12% Tris-Tricine Ready Gel (Bio-Rad Laboratories Inc., Hercules, CA, USA) and then electrotransferred to a Hybond-enhanced chemiluminescence membrane (Amersham). The membranes were incubated overnight at 4°C with rabbit polyclonal antibody against TIPE (1:4,500; Abcam plc, Cambridge, UK) and mouse monoclonal antibody against β-actin (1:5,000; Sigma-Aldrich Co., St Louis, MO, USA). The bands were detected by using an enhanced chemiluminescence detection system, where β-actin served as the control. Scanning densitometry was performed using Adobe Photoshop (Apple, Inc, Cupertino, CA, USA).
6
Western Blot Analysis of LC3B in L-02 Cells
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L-02 cells in each 25-cm2 culture flask were collected and proteins were extracted with 1 ml mixture of RIPA lysis buffer (P0013B; Beyotime Institute of Biotechnology, Haimen, China) and phenylmethylsulfonyl fluoride (final concentration, 1 mmol/l). Protein concentration was determined using a bicinchoninic acid protein assay kit (P0012; Beyotime Institute of Biotechnology). Thirty micrograms of protein samples were separated by SDS-PAGE and then electro-transferred onto the polyvinylidene difluoride membranes (Millipore Corporation, Billerica, MA, USA). Membranes were blocked for 1 h with 5% skimmed milk in TBST buffer [50 mmol/l Tris (pH 7.6), 150 mmol/l NaCl and 0.1% Tween-20] and incubated with rabbit monoclonal antibodies against human LC3B (Cell Signaling Technology, Inc., Beverly, MA, USA) overnight at 4°C. β-actin was used as an internal control and was detected using a mouse monoclonal antibody against β-actin (Sigma, St. Louis, MO, USA). Following incubation with secondary antibodies, enhanced chemiluminescence detection reagents (Beyotime Institute of Biotechnology) were used to detect the signals. The intensity of the signals was quantified with Image Lab 3.0 software (Bio-Rad Laboratories, Inc.).
7
Immunoblotting and Immunohistochemistry Antibody Protocol
8
Quantification of Airway Epithelial ANO1
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Sectioned membranes of ALI cultures were added to RIPA (Radioimmunoprecipitation assay) buffer (Sigma-Aldrich, USA) supplemented with protease inhibitor cocktail on ice for 1 h and centrifuged at 12,000×g for 10 min at 4 °C. Total protein extracted was quantified using BCA assay kit (Pierce, Thermo Fisher Sci, USA) on the spectrophotometer (BioTek, USA) at 450 nm. Equal concentrations (20 µg) of protein were used for SDS-PAGE based protein separation on 4–12% gels (Bio-Rad) and transferred on a PVDF membrane (Millipore, Sigma, USA). Membranes were blocked using Everyblot (BioRad, USA) for 20 min at room temperature, incubated with primary rabbit raised polyclonal antibody against ANO1 (1:1000; Invitrogen, Thermo Fisher Scientific, USA), mouse monoclonal antibody against β-actin (1:500; Sigma-Aldrich, USA) overnight at 4 °C. Membranes were washed with TBST for 5 min and incubated with respective HRP-conjugated anti-rabbit and anti-mouse secondary antibody (Sigma-Aldrich, USA) for 2 h at room temperature. Protein detection was done using the ECL detection system (Thermo Fisher Sci, USA) and relative quantification for band intensity was done using ImageJ software.
9
Quantification of Protein Levels
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Protein levels of CORO1C, cyclin D1 and vimentin were determined by western blot essentially as described earlier 12 , 13 , 16 . CORO1C rabbit polyclonal antibody, cyclin D1 rabbit polyclonal antibody, vimentin rabbit polyclonal antibody (all 1 : 1000; Proteintech Group), and mouse monoclonal antibody against β‐actin (1 : 5000; Sigma‐Aldrich, St. Louis, MO, USA) were used.
10
Mitochondrial Complex Protein Analysis
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Cortical homogenates containing 20 μg of protein were loaded on a 4–20% SDS-PAGE gel (BioRad) and transferred onto PVDF membranes (Millipore). They were then incubated with an antibody cocktail targeting one representative subunit of each of the five mitochondrial complexes (MitoSciences, complex I subunit NDUFB8, complex II-30 Da, complex III Core protein 2, complex IV subunit I, complex V alpha subunit), and mouse anti-mitofusin2 (Mfn2) (Abcam). Immunoreactive protein was detected using an enhanced chemiluminescence reagent (ECL Clarity, Bio Rad). Densitometric quantification was carried out with ImageQuant TL Image Analysis software version 7.0 (LAS 4000, GE Healthcare). Protein loading was monitored using a mouse monoclonal antibody against β-actin (Sigma-Aldrich).
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