Total protein was first separated by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes (Millipore, USA). The membranes were blocked with 5% nonfat milk and incubated with a rabbit anti-TSPAN1 polyclonal antibody at a dilution of 1:1000 (Abcam, USA) or a mouse anti-beta-actin monoclonal antibody at a dilution of 1:2,000 (Sigma, USA). The membranes were subsequently incubated with a goat anti-mouse or anti-rabbit horseradish peroxidase secondary antibody (Sigma, USA). The protein complex was detected using enhanced chemiluminescence reagents (Pierce, France). Beta-actin was used as an internal control.
Mouse anti beta actin monoclonal antibody
Manufactured by Merck Group
Sourced in United States
The Mouse anti-beta-actin monoclonal antibody is a laboratory reagent used for the detection and quantification of the beta-actin protein in biological samples. Beta-actin is a highly conserved cytoskeletal protein found in all eukaryotic cells and is commonly used as a loading control or reference protein in various biochemical and cell biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Merck Group (2 mentions)
Goat anti mouse horseradish peroxidase secondary antibody, by Merck Group (2 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (2 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (2 mentions)
Goat anti mouse or anti rabbit horseradish peroxidase secondary antibody, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
X ray film, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico substrate, by Thermo Fisher Scientific (1 mentions)
Rabbit anti at1 and at2 receptor polyclonal antibodies, by Santa Cruz Biotechnology (1 mentions)
Mouse anti tgf β1 monoclonal antibody, by Abcam (1 mentions)
Mouse monoclonal anti myc antibody, by Cell Signaling Technology (1 mentions)
Anti tubulin b512 mouse monoclonal antibody, by Merck Group (1 mentions)
Anti rala mouse monoclonal antibody, by BD (1 mentions)
Hrp conjugated anti mouse secondary antibody, by GE Healthcare (1 mentions)
Rabbit polyclonal anti gfp, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (1 mentions)
Polyvinylidene fluoride transfer membrane, by Merck Group (1 mentions)
Donkey anti rabbit igg hrp, by Jackson ImmunoResearch (1 mentions)
14 protocols using mouse anti beta actin monoclonal antibody
1
Western Blot Analysis of TSPAN1 Expression
2
Western Blotting Analysis of DNAJB13 in Mouse Tissues
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The protein samples were analyzed by western blotting with antibodies against DNAJB13 and beta-actin, respectively. The multi-tissue proteins from adult BALB/c mice and the testes proteins from different stages of BALB/c mice were separated by SDS–PAGE on a 12% polyacrylamide gel and transferred to PVDF membrane (Millipore). Western blotting analysis was performed as described previously [13 (link)]. The membranes were blocked with TBST (25 mM Tris–HCl, pH 8.0, 125 mM NaCl, 0.1% Tween 20) containing 5% nonfat dry milk for 4 h at r.t. and incubated with the primary antibodies in blocking buffer overnight at 4°C. After washing with TBST, the blots were incubated with the secondary antibodies in blocking buffer for 1 h at r.t. Both antibodies were conjugated to horseradish peroxidase. The membranes were detected with Lumigen™ PS-3 detection kit (GE Healthcare UK Limited) according to the manufacturer’s directions and exposed to X-ray film (Fuji, Tokyo, Japan). The primary antibodies used in this study were TSARG6 (F-20) goat polyclonal antibody (1:50 dilution) (Santa Cruz), and mouse anti-beta-actin monoclonal antibody (1:1000 dilution) (Sigma). The secondary antibodies were HRP-conjugated donkey anti-goat IgG antibody (1:20000 dilution) (KPL) and HRP-conjugated goat anti-mouse IgG antibody (1:4000 dilution) (KPL).
3
Western Blot Analysis of Cellular Proteins
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Cellular proteins were extracted and separated in SDS-PAGE gels, first separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes (Bio-Rad Laboratories, USA). The membranes were blocked with 5% nonfat milk and incubated with a mouse anti-AMFR polyclonal antibody at a dilution of 1:500 (Abcam, USA), a rabbit anti-activated NOTCH1 antibody (Abcam, USA), or a mouse anti-beta-actin monoclonal antibody at a dilution of 1:1000 (Sigma, USA). The membranes were subsequently incubated with a goat anti-mouse horseradish peroxidase secondary antibody (Sigma, USA). The protein complex was detected using enhanced chemiluminescence reagents (Pierce, France).
4
Western Blot Protein Analysis
5
Quantifying Protein Expression in Tissue
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Freshly frozen transmural tissue samples obtained from the different groups were homogenized in ice-cold lysis buffer. Protein concentration was measured by the DC protein assay method as previously reported.4 (link) In brief, proteins (60 μg) were separated by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified with the following antibodies to quantify their protein levels: rabbit anti-AT1 and AT2 receptor polyclonal antibodies (Santa Cruz Biotechnology Inc, Dallas, TX, USA), a mouse anti-TGFβ1 monoclonal antibody (Abcam, Cambridge, UK), rabbit anti-Smad2, 3, 4, and 7 monoclonal antibodies (Cell Signaling Technology, Danvers, MA, USA), a mouse anti-collagen type III monoclonal antibody (Sigma-Aldrich Co, St Louis, MO, USA), and a mouse anti-beta actin monoclonal antibody (Sigma-Aldrich Co), respectively. After incubation with the primary antibody, the bound antibody was visualized with respective horseradish peroxidase-coupled secondary antibody. The membrane was then incubated with chemiluminescence developing agents and exposed to an X-ray film. The actin level was determined in every sample. Films were scanned and band densities were quantified with densitometric analysis using the ImageJ system (NIH). The final band density was normalized with actin.
6
Western Blot Analysis of PLD1 Protein
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Harvested proteins were first separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes (Millipore, USA). The membranes were blocked with 5% nonfat milk and incubated with a mouse anti-PLD1 polyclonal antibody at a dilution of 1:500 (Cell signal, USA) or a mouse anti-beta-actin monoclonal antibody at a dilution of 1:500 (Sigma, USA). The membranes were subsequently incubated with a goat anti-mouse horseradish peroxidase secondary antibody (Sigma, USA). The protein complex was detected using enhanced chemiluminescence reagents (Pierce, France). Endogenous beta-actin was used as the internal control.
7
Overexpression of SOX11 in HEK293T Cells
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HEK293T cells were seeded into 6-well plate and transfected with the indicated SOX11 constructs. 24 h after transfection, cells were washed with ice-cold 1x PBS and lysed with SDS lysis buffer (100 mM pH6.8 Tris-HCl, 10% Glycerol, 1% SDS). Whole cell lysates were separated on 10% SDS-PAGE gels, transferred to polyvinylidene difluoride (PVDF) membranes. After blocking in 5% non-fat milk in TBS-T for 1 h, membranes were then incubated overnight at 4°C with mouse anti-Myc monoclonal antibody (Cell Signaling Technology) and mouse anti-beta-Actin monoclonal antibody (Sigma-Aldrich). Proteins were detected using a chemiluminescence system with a horseradish peroxidase conjugated secondary antibody.
8
Cisplatin Cytotoxicity Mechanism Elucidation
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Cells were seeded in 6-well plates (7 × 105 cells/well), incubated for 24 h and then exposed to 10 µM cisplatin for 6 h. Cells were harvested and proteins were isolated as described previously (Baier et al., 2022 (link)). Polyvinylidene difluoride membranes were incubated for 18 h while shaking at 4°C in TBS/0.1% Tween/3% BSA containing the following primary antibodies: anti-CTR1 rabbit polyclonal antibody (#sc-66847, 1:500), purchased from Santa Cruz Biotechnology (Dallas, TX, United States) and anti-beta-Actin mouse monoclonal antibody (#A5441, 1:2,000), purchased from Sigma-Aldrich. The membranes were then incubated for 1 h at RT in TBS/0.1% Tween/1% BSA containing the following secondary antibodies: anti-rabbit IgG HRP-linked antibody (#7074, 1:5,000), purchased from Cell Signaling Technology (Danvers, MA, United States) or goat anti-mouse-IgG (Fc specific)-peroxidase antibody (#A0168, 1:10,000), purchased from Sigma-Aldrich.
9
Quantitative Immunoblotting Analysis of Cellular Proteins
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Whole-cell extracts were prepared from HeLa cells transfected with control siRNA, TD-60 siRNA or RalA siRNA. Immunoblotting analysis was performed using the following primary antibodies: anti-RCC2 rabbit polyclonal antibody (Cell Signalling 1:100–1:200), anti-RalA mouse monoclonal antibody (BD Bioscience 1:100), anti-Ran mouse monoclonal antibody (BD Bioscience 1:1,000), anti-Tubulin B512 mouse monoclonal antibody (Sigma 1:10,000), anti-Beta Actin mouse monoclonal antibody (Sigma 1:1,000) and anti-GFP rabbit polyclonal (Life Technology, 1 μg ml−1). For protein detection and quantitation, we used donkey IRDye 800 secondary antibodies (LI-COR Bioscience 1:10,000), HRP-conjugated anti-mouse secondary antibody (GE Amersham 1:5,000) and anti-rabbit IgG, HRP-linked Antibody (Cell Signalling 1:3,000). Uncropped versions of western blots can be found in Supplementary Fig. 9 .
10
Evaluating c-KIT Mutant Activation
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To evaluate the activation status of the mutant c-KIT, each cell line was seeded in 6-well plates at 5.0 × 105 cells/well and incubated for 3 h in the plain RPMI. After incubation, cells were stimulated with fSCF (100 ng/ml) for 5 min and harvested and lysed with NP40 lysis buffer [1% NP 40, 10 mM Tris HCL (pH 7.5), 150 mM NaCl, 1 mM EDTA, protease inhibitor cocktails (Nacalai Tesque, Kyoto, Japan), 1 mM Na3VO4, and 50 mM NaF]. The resulting protein lysates were loaded on SDS–polyacrylamide gels and electrophoresed followed by blotting to polyvinylidene fluoride transfer membrane (Merck, Darmstadt, Germany). Then, the membrane was blocked with 5% skim milk blocking buffer, followed by incubation with the primary antibody, rabbit polyclonal anti-phospho-c-KIT antibody (Tyr719) (Cell Signaling Technology, Danvers, MA, USA), goat polyclonal anti-c-KIT antibody (M-14, Santa Cruz Biotechnology), or mouse monoclonal anti-beta actin antibody (Sigma-Aldrich). Horseradish peroxidase (HRP)-conjugated antibodies, including donkey anti-rabbit IgG-HRP (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), goat anti-mouse IgG-HRP (Bio-Rad Laboratories) and goat anti-mouse IgG-HRP (Bio-Rad Laboratories) were used as secondary antibodies. The specific proteins were detected using the ECL reagent (PerkinElmer, Waltham, MA, USA).
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