Oocytes extracts were prepared as previously described (White et al., 1992 (link)), separated on 12% SDS gels and transferred to nitrocellulose membranes. Blots were blocked with 5% milk 0.1% Tween20 in TBS, probed with polyclonal antibodies against Cx26 or Cx43 (Life Technologies, Carlsbad CA), followed by horseradish peroxidase conjugated secondary antibodies (Jackson Laboratories and GE Healthcare). A monoclonal β-actin antibody (Abcam, Cambridge, MA) was used as a loading control. Band intensities were quantified using ImageJ software. The phosphorylated and non-phosphorylated forms of Cx43 (two bands) were quantified and expressed as a single value.
β actin monoclonal antibody
Manufactured by Abcam
Sourced in United Kingdom
β-actin monoclonal antibody is a primary antibody that specifically recognizes the β-actin protein, a highly conserved cytoskeletal protein found in eukaryotic cells. This antibody can be used for the detection and quantification of β-actin in various applications, such as Western blotting, immunohistochemistry, and immunofluorescence.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (3 mentions)
Polyclonal antibodies against cx26 or cx43, by Thermo Fisher Scientific (2 mentions)
Transforming growth factor β1 tgf β1 antibody, by Abcam (1 mentions)
Real time pcr reagents, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Gemini Bio (1 mentions)
Secondary peroxidase linked whole antibodies, by GE Healthcare (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Iblot device, by Thermo Fisher Scientific (1 mentions)
Visionworks ls software, by Analytik Jena (1 mentions)
Doxorubicin, by Merck Group (1 mentions)
Docetaxel, by Merck Group (1 mentions)
Puromycin, by Selleck Chemicals (1 mentions)
Mk571, by Selleck Chemicals (1 mentions)
Dulbecco s modified eagle medium dmem, by PAN Biotech (1 mentions)
Paclitaxel, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by PAN Biotech (1 mentions)
P akt, by Abcam (1 mentions)
Phosphorylated p65, by Cell Signaling Technology (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
12 protocols using β actin monoclonal antibody
1
Western Blot Analysis of Connexin Proteins
2
Western Blot Analysis of Connexin Proteins
3
Western Blotting of TGF-β1 in Tissue Homogenates
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CC tissue homogenates were used for Western blotting performed according to standard procedures. Briefly, homogenates were lysed in lysis buffer containing 50mM Tris–HCl (pH 8.0). These homogenates were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred onto a polyvinylidene fluoride membrane blocked with 5% skim milk, and hybridized with primary antibodies. Transforming growth factor-β1 (TGF-β1) antibody and monoclonal β-actin antibody were purchased from Abcam. After incubation with horseradish-peroxidase-conjugated secondary antibody at room temperature, immunoreactive proteins were detected using a chemiluminescent electrogenerated chemiluminescence assay kit (Abcam) according to the manufacturer's instructions.
4
Isolation and Osteogenic Potential of Human ASCs
5
Western Blot Analysis of Connexin 26
6
Characterization of RMS Cell Lines
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Protein expression of all RMS cell lines was also characterized by WB assay, and for those cell lines that resulted IHC ALK positive, its phosphorylated form was also investigated. Samples containing 200 μg of protein per lane were separated on precast 4–12% NuPAGE bis-tris gels (Invitrogen), and were transferred onto Hybond ECL nitrocellulose membranes (Invitrogen) using the NuPAGE transfer buffer and iBlot device (Invitrogen). Nitrocellulose membranes were blocked in PBS-Tween 20 with 5% skim milk, first incubated with the primary antibodies (Cell Signaling) and then with the secondary peroxidase linked whole antibodies (GE Healthcare Europe). Bound antibodies were detected using the Super Signal chemiluminescent substrate (GE Healthcare Europe). β-Actin monoclonal antibody (Abcam) was used to confirm equal protein loading on the gel. Filters were autoradiographed, and autoradiographs were scanned and quantified by densitometric analysis using Vision Works LS software (UVP, Upland, CA, USA). The H2228 lung adenocarcinoma cell line, positive for ALK rearrangement (EML4-ALK), was utilized as positive control for ALK expression (total protein and phosphorylated form at 80kDa).
7
Transporter-Mediated Paclitaxel Resistance Study
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Paclitaxel, docetaxel, and doxorubicin were purchased from Sigma-Aldrich (St Louis, MO, USA). MK-571 and puromycin were obtained from Selleck (Shanghai, China). All drugs were dissolved in DMSO. Cell Counting Kit-8 (CCK8) was purchased from Pepro Tech (Wuhan, China). Pig kidney cells (LLC-PK1) were purchased from Wuhan Bafeier Biological Co. Ltd (Wuhan, China). LLC-PK1 stable transfected with empty vector (pcDNA3.1), ABCC21249G wild-type allele and ABCC21249A variant allele were obtained from Biofavor Biotech (Wuhan, China). Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from PAN-Biotech (Aidenbach, Germany). MRP2 and β-actin monoclonal antibody were purchased from Abcam (Cambridge, UK). HRP-conjugated IgG was also obtained from Abcam (Cambridge, UK). BCA protein assay kit was obtained from Innova Biosciences (Cambridge, UK).
8
Comprehensive Western Blotting Protocol for Cellular Signaling Analysis
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Western blotting was performed as described previously (21 (link)). The following antibodies were used: Rabbit antibodies against HNF-4α (cat. no. ab201460; Abcam), E-cadherin (cat. no. 24E10; Cell Signaling Technology, Inc.), N-cadherin (cat. no. 13116; Cell Signaling Technology, Inc.), β-catenin (cat. no. 8480; Cell Signaling Technology, Inc.), phosphorylated (P)-p65 (cat. no. 3033; Cell Signaling Technology, Inc.), p65 (cat. no. 8242; Cell Signaling Technology, Inc.), P-STAT3 (cat. no. ab76315; Abcam), P-AKT (cat. no. ab38449; Abcam), AKT (cat. no. ab8805; Abcam) and β-actin monoclonal antibody (cat. no. HRP-60008; Proteintech Group, Inc.); mouse antibodies against STAT3 (cat. no. ab119352; Abcam).
9
Quantitative Protein Expression Analysis
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According to the method of Kang et al. [13 (link)], homogenates of medaka gills or embryos were prepared. Aliquots containing 20 μg of homogenates were heated at 65°C for 25 min and fractionated by electrophoresis on SDS-containing 7.5% polyacrylamide gels. Transferred PVDF blots were incubated with VILL polyclonal antibody (1:200,000 dilution) or β-actin monoclonal antibody (1:10,000 dilution; 8226, Abcam, Cambridge, UK) for 2 hrs, followed by a 1-hr reaction with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG (Pierce, Rockford, IL, USA). Blots were developed using the SuperSignal West Pico Detection Kit (#34082, Pierce) and analyzed with Image Lab software version 3.0 (Bio-Rad) after densitometric scanning of membranes using a ChemiDoc XRS+ (Bio-Rad). Results were converted to numerical values to compare relative intensities of immunoreactive bands.
10
Protective Effects of Laminarin on RAECs
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The materials used in the present study included RAECs (Jiangsu Chi Scientific Co., Ltd.), complete Dulbecco's modified Eagle's medium (DMEM) containing FBS (both 3-7202; Jiangsu Chi Scientific Co., Ltd.), a Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.), LBPs (Ningxia Qiyuan Pharmaceutical, Co., Ltd.), anti-B-cell lymphoma 2 (Bcl-2) antibody (cat. no. ab59348; Abcam), anti-Bcl-2-associated X protein (Bax) antibody [E63] (cat. no. ab32503; Abcam), and β-actin monoclonal antibody (cat. no. TA811000), horseradish peroxidase (HRP)-goat anti-rat immunoglobulin G (IgG) (cat. no. TA130038) and HRP-goat anti-rabbit IgG (cat. no. TA130023) all from OriGene Technologies, Inc. A total protein extraction kit (KGP250) and bicinchoninic acid assay (BCA) kit (KGP902; Nanjing KeyGen Biotech. Co., Ltd.), a SOD assay kit-WST-1, a Microscale MDA assay kit (thiobarbituric acid method) and a nitric oxide (NO) assay kit (A012-1-2; Colorimetric; Nanjing Jiancheng Bio-Engineering Institute Co., Ltd.) were also used. The major instruments included electrophoresis apparatus, a Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad Laboratories, Inc.), a light microscope (Olympus Corporation), an Amersham Imager 600 (GE Healthcare Life Sciences and a CO2 incubator (Heraeus).
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