Anti human β actin

Manufactured by Cell Signaling Technology

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Anti-human β-actin is a primary antibody that specifically binds to the β-actin protein, a widely-expressed structural protein found in eukaryotic cells. It can be used to detect and quantify β-actin levels as a control for protein expression studies.

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β-actin (6244 citations) Anti-β-actin (1878 citations) Anti-actin (219 citations) Mouse anti-human β-actin (20 citations) Human β-actin (6 citations) Mouse anti-human β-actin monoclonal antibody (6 citations) Rabbit anti-human β-actin monoclonal antibody (5 citations) Anti-human β-actin antibody (5 citations) Mouse anti-human beta-actin (β-actin) antibody (3 citations) Mouse anti-human β-actin antibody (3 citations)

8 protocols using anti human β actin

Characterization of Anti-LiMTAP Antibody

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Four putative antigenic LiMTAP peptides were predicted using Antigenic, a method described previously [50 (link)], implemented in EMBOSS. The peptide (AIVTKPEHIPAETKQRIAPLVASK), located in the C-terminus of LiMTAP, was used to produce a polyclonal antibody (Genescript, USA). The specificity of this anti-LiMTAP was assessed by western blot.
PBMC lysates were used as human control in western blot. Three amounts (3 μg, 7 μg and 15 μg) of LV50 lysates and 15 μg of PBMC lysates were resolved by electrophoresis on SDS-polyacrylamide gels (12%), transferred onto a polyvinylidene difluoride (PVDF) blotting membrane (GE Healthcare life sciences, Amersham, Germany) and probed by immunoblotting with anti-LiMTAP (d: 1/10000) (Genescript, USA) and anti-horseradish peroxidase (HRP) conjugated secondary (d: 1/5000) antibodies (Promega, Madisson WI). The proteins were visualized by the enhanced chemiluminescence detection system (ECL, Pierce, Rockford, IL). The same experiment was assessed using an anti-human β-actin (d: 1/5000) (Cell Signaling Technology, Danvers, MA) as a positive control for human PBMC.

Abid H., Harigua-Souiai E., Mejri T., Barhoumi M, & Guizani I. (2017). Leishmania infantum 5’-Methylthioadenosine Phosphorylase presents relevant structural divergence to constitute a potential drug target. BMC Structural Biology, 17, 9.

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Western Blot Analysis of Cellular Proteins

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Total cellular proteins were solubilized in RIPA lysis buffer containing protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA) and 1 mM PMSF. Proteins were loaded on a 10% SDS gel, resolved using SDS-PAGE and subsequently transferred electrophoretically to a PVDF membrane (EMD Millipore). The membranes were blocked in 5% non-fat-dried milk at room temperature for 30–60 min, and subsequently incubated with primary antibodies overnight at 4°C at the following dilutions: GAPDH (Santa Cruz Biotechnology, Inc.; cat. no. sc-25778) at 1:1,000, Ago2 (Abcam; cat. no. ab57113) at 1:2,000, STAT3 (Santa Cruz Biotechnology, Inc. cat. no. sc-8019) at 1:300, anti-human β-actin (Cell Signaling Technology, Inc.; cat. no. 4970) at 1:1,000. Subsequently, the membranes were washed and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Inc.; cat. no. 7074) at a dilution of 1:2,000. Following several washes, signals were visualized using an ECL system (Tiangen Biotech Co., Ltd.). β-actin was used as the loading control. Western blot protein grayscale values were detected and analyzed by ImageJ (1.48V; NIH).

Liu P., Zhong Y., Cao T., Sheng X, & Huang H. (2020). A frequent somatic mutation in the 3′UTR of GAPDH facilitates the development of ovarian cancer by creating a miR-125b binding site. Oncology Reports, 44(3), 887-896.

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Western Blot Analysis of Apoptosis-Related Proteins

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After 6 hours of treatment, Jurkat cells were pellet at 1800 rpm for 10 minutes. Then, cells were suspended in a hot 2 x SDS gel sample buffer following the manufacturer’s conditions. The extracts were separated in 15% SDS-PAGE, then electrotransferred to Immun-BlotTM PVDF Membrane (Bio-rad). The membranes were blocked with BSA 1% for 1 hour at room temperature and incubated overnight with the primary antibody at appropriate dilutions. After being washed, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies, and immunoreactive bands were detected SuperSignal^® West Pico Chemiluminescent Substrate (PIERCE, Thermo Scientific). Cells were stained with anti-human BID (R&D system), anti-Human β-Actin (Cell signaling), anti-Human Bax and anti-Human Bcl-2 (BD Bioscience), mouse anti-goat, Goat-anti Rabbit, and Goat-anti-mouse (Santa Cruz Biotechnology) and subjected to western blot analysis.

Duarte A., Montagna D.R., Pastorini M, & Alemán M. (2023). Apoptosis-mediated inhibition of human T-cell acute lymphoblastic leukemia upon treatment with Staphylococus Aureus enterotoxin-superantigen. Frontiers in Immunology, 14, 1176432.

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Subcellular Fractionation and Western Blotting

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Nuclear and cytoplasmic fractions were isolated using NE-PER ^® Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Rockford, IL, USA). Western blotting was carried out by standard procedures as previously described. ^{[13 (link)]} The primary antibodies used for the assay were as follows: anti-human SEMA3F (1:1000) (Chemicon, Temecula, CA, USA), anti-human MST2, MST1, MOB1, p-MOB 1, LATS1/2, YAP/TAZ, cleaved caspase-3, BCL2, p-YAP Ser397, p-YAP Ser127, and CCND2 (1:500) (Cell Signaling Technology, Danvers, MA, USA), anti-human β-actin (1:1000) (Cell Signaling Technology), anti-human CD20 (1:1000) (Abcam Cambridge, MA, USA), anti-human TAZ (1:1000) (Abcam), and anti-human Lamin B1 (1:1000) (Novus Biologicals, Littleton, CO, USA).

Li Q., Ma N., Li X., Yang C., Zhang W., Xiong J., Zhu L., Li J., Wen Q., Gao L., Yang C., Rao L., Gao L., Zhang X, & Rao J. (2023). Reverse effect of Semaphorin-3F on rituximab resistance in diffuse large B-cell lymphoma via the Hippo pathway. Chinese Medical Journal, 136(12), 1448-1458.

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Immunoblotting of IRG1 in THP-1 Cells

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THP-1 cells were lysed in 1xLysis Buffer (Cell Signaling, Danvers, MA) supplemented with protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Protein concentration was assayed using the BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). Equal amounts of protein from each sample were subjected to SDS-PAGE, followed by immunoblotting with anti-human IRG1 (Cell Signaling, Danvers, MA) or anti-human β-actin (Cell Signaling, Danvers, MA) antibodies. Immunoblots were visualized with the supersignal substrate system (Thermo Fisher Scientific, Waltham, MA), and chemiluminescence was captured with Kodak X-Omat XLS-1 film.

Zhu X., Long D., Zabalawi M., Ingram B., Yoza B.K., Stacpoole P.W, & McCall C.E. (2020). Stimulating pyruvate dehydrogenase complex reduces itaconate levels and enhances TCA cycle anabolic bioenergetics in acutely inflamed monocytes. Journal of leukocyte biology, 107(3), 467-484.

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Western Blot Analysis of STIP1 Expression

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To measure STIP1 expression, cells were lysed in PRO-PRE Protein Extraction Solution (Intron Biotechnology, Seongnam, Korea). Cell lysates (100 μg of total protein) were separated in 10% acrylamide gels by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk in TBST (50 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.5) for 1 h at room temperature, followed by incubation with anti-STIP1 rabbit polyclonal antibody (mouse monoclonal, Cat.# sc-32761, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Smad5 (Cell Signaling, Danver, MA), anti-phosphoSmad1/5(Ser463/465) (Cell Signaling), anti-human β-actin (Cell Signaling), or anti-GAPDH (Santa Cruz Biotechnology) overnight at 4°C. The membranes were then incubated with secondary antibodies conjugated with horseradish peroxidase (GE Healthcare, Munich, Germany) and visualized on AGFA X-ray film (Agfa Health Care, Mortsel, Belgium) using the SuperSignal Chemiluminescence kit (Thermo Scientific, Rockford, IL).

Cho H., Kim S., Shin H.Y., Chung E.J., Kitano H., Park J.H., Park L., Chung J.Y., Hewitt S.M, & Kim J.H. (2014). Expression of Stress-Induced Phosphoprotein1 (STIP1) is Associated with Tumor Progression and Poor Prognosis in Epithelial Ovarian Cancer. Genes, chromosomes & cancer, 53(4), 277-288.

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Evaluating Signaling Pathways in Pre-B ALL

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For Western blot analysis, 30 μg of total protein extracts prepared from NALM6 or primary pre-B ALL cells from SP of PDX mice were separated on SDS-PAGE and transferred to a PVDF membrane (Bio-Rad, Hercules, CA, USA). The following commercially available antibodies and dilutions were used for Western blotting: human anti-STAT3 (ab119352, Abcam, UK), human anti-phospho-STAT3 (Y705) (ab76315, Abcam, UK), human anti-SRC (ab32102, Abcam, UK), human anti-phospho-SRC (Y419) (ab185617, Abcam, UK), human anti-RB (9313, Cell Signaling Technology, MA, USA), human anti-phospho–RB (Ser807/811) (8516, Cell Signaling Technology, MA, USA), human anti-C-myc (13987, Cell Signaling Technology, MA, USA), anti-AKT (pan) (4691P, Cell Signaling Technology, MA, USA), anti-phospho-AKT (Ser473) (4060P, Cell Signaling Technology, MA, USA), anti-IRS-1 (2390S, Cell Signaling Technology, MA, USA), and human anti–β-actin (4970, Cell Signaling Technology, MA, USA). Western blot analysis was performed with cell lines or primary pre-B ALL cells from SP following GZD824 treatment, and signal intensities were normalized to β-actin signals.

Ye W., Jiang Z., Lu X., Ren X., Deng M., Lin S., Xiao Y., Lin S., Wang S., Li B., Zheng Y., Lai P., Weng J., Wu D., Ma Y., Chen X., Wen Z., Chen Y., Feng X., Li Y., Liu P., Du X., Pei D., Yao Y., Xu B., Ding K, & Li P. (2016). GZD824 suppresses the growth of human B cell precursor acute lymphoblastic leukemia cells by inhibiting the SRC kinase and PI3K/AKT pathways. Oncotarget, 8(50), 87002-87015.

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Evaluation of BIIB021 Cytotoxicity in SKM-1 Cells

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SKM-1 cells (JCRB0118; Japanese Collection of Research Bioresources Cell Bank, Osaka, Japan) were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (Gibco) at 37°C in a humidified atmosphere of 5% CO₂. BIIB021 was purchased from Selleck Chemicals (Houston, TX, USA). Methylcellulose and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All antibodies used in the western blot analysis were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA), with the exception of human anti-β-actin. Insulin-like growth factor-1 (IGF-1) was purchased from Peprotech (Rocky Hill, NJ, USA). The caspase inhibitors z-IETD-fmk and z-LEHD-fmk were purchased from Biovision (Palo Alto, CA, USA).

LIN S., LI J., ZHOU W., QIAN W., WANG B, & CHEN Z. (2014). BIIB021, an Hsp90 inhibitor, effectively kills a myelodysplastic syndrome cell line via the activation of caspases and inhibition of PI3K/Akt and NF-κB pathway proteins. Experimental and Therapeutic Medicine, 7(6), 1539-1544.

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