β actin a5316

Manufactured by Merck Group

Sourced in United States

β-actin (A5316) is a purified protein that is commonly used as a reference or control in various laboratory applications. It is a highly conserved cytoskeletal protein that is involved in cell structure and motility. The product is available in a lyophilized powder form.

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β-actin (6886 citations) A5316 (203 citations) Anti-β-actin (A5316) (29 citations) Anti-β-actin antibody (A5316) (17 citations) β-actin antibody (A5316) (13 citations) Mouse anti-β-actin (A5316; (8 citations) Anti‐β‐actin mouse monoclonal antibody (A5316 (6 citations) Mouse monoclonal anti-β-actin (A5316, (3 citations) β-Actin mAb (A5316) (2 citations) Antibody against β-actin (A5316, (2 citations)

37 protocols using β actin a5316

Optimizing Metabolic Signaling in Cell Studies

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Phospho-AMPKα (Thr172) (2535), AMPKα (2532), LKB1 (3050), phospho-Akt (Ser-473) (4058) and pan-Akt (4691) antibodies were from Cell Signaling Technology (Danvers, MA). NTR1 antibody (sc-374492) was from Santa Cruz Biotechnology (Dallas, TX). CaMKK2 (ab168818), NTR3 (ab16640), and F4/80 (ab100790) antibodies were from Abcam (Cambridge, MA). FLAG (F1804) and β-actin (A5316) antibodies were from Sigma-Aldrich (St. Louis, MO). AICAR was from Cayman (Ann Arbor, MI). Oleate sodium, NT_1–13, glucose and human insulin were from Sigma. Deuterated oleic acid (CLM-460-PK) was obtained from Cambridge Isotope Laboratories (Tewksbury, MA). SR 48692 was from Tocris (Minneapolis, MN). Lipofectamine® RNAiMAX and LTX Reagent with PLUS^™ transfection reagents and Trizol were from Life Technologies (Grand Island, NY). pSG5-FLAG-CaMKK2 rat FL was a gift from Anthony Means^{30 (link)} (Addgene plasmid #32449, Cambridge, MA). Primers for RT-PCR were from Integrated DNA Technologies (Coralville, IA).

Li J., Song J., Zaytseva Y.Y., Liu Y., Rychahou P., Jiang K., Starr M.E., Kim J.T., Harris J.W., Yiannikouris F.B., Katz W.S., Nilsson P.M., Orho-Melander M., Chen J., Zhu H., Fahrenholz T., Higashi R.M., Gao T., Morris A.J., Cassis L.A., Fan T.W., Weiss H.L., Dobner P.R., Melander O., Jia J, & Evers B.M. (2016). An obligatory role for neurotensin in high fat diet-induced obesity. Nature, 533(7603), 411-415.

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Metabolic Reprogramming in Lung Cancer

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The normal human bronchial epithelioid cell (HBE) and the non-small-cell lung cancer cell lines including NCI-H1650, NCI-H1299, A549, NCI-H460, HCC4006, NCI-H1975, and NCI-H358 were obtained from ATCC and cultured with recommended culture medium. The primary antibodies for western blotting including anti-TRAF6 (#8028), hexokinase-1 (#2024), hexokinase-2 (#2867), GLUT1 (#12939), PKM2 (#4053), LDHA (#35 82), VDAC-1 (#4661), phosphor-Akt (#4060), Akt (#8596), phosphor-S6 (#4858), and ubiquitin (#58395) as well as the secondary anti-rabbit IgG HRP (#7074) were products of Cell Signaling Technology Inc. (Danvers, MA). β-Actin (A5316) was obtained from Sigma-Aldrich. In immunohistochemistry staining, the primary antibodies against hexokinase-2 (ab227198) and Ki67 (ab15580) were products of Abcam. TRAF6 shRNA#1 (TRCN0000007350) and shRNA#2 (TRCN0000007351) were purchased from the Sigma Mission shRNA library. The constitutively active Akt (CA-Akt) plasmid (Cat. #10841) and pLKO.1 GFP shRNA (Cat. #30323) were purchased from Addgene (Cambridge, MA, USA). Recombinant human insulin-like growth factor 1 (IGF-1) was a product of R&D (Cat. 291-G1-200). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA).

Feng L., Feng S., Nie Z., Deng Y., Xuan Y., Chen X., Lu Y., Liang L, & Chen Y. (2021). TRAF6 Promoted Tumor Glycolysis in Non-Small-Cell Lung Cancer by Activating the Akt-HIFα Pathway. BioMed Research International, 2021, 3431245.

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Exosome Molecular Profiling by Western Blot

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Cells and exosomes were lysed in lysis buffer [50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 100μg/ml phenylmethylsulphonyl fluoride, 0.5 μg/ml leupeptin, and 1 μg/ml aprotinin]. Protein concentrations were determined using a Micro BCA™ protein assay kit (Pierce Biotechnology). Equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to PVDF membranes. Membranes were blocked with 5% skim milk in TBS containing 0.1% Tween-20 for 2 h at room temperature and incubated with the appropriate primary and secondary antibodies. Antibodies against CD63 (HPA010088, 1/1000) and β-actin (A 5316, 1/10000) were purchased from Sigma-Aldrich. Antibodies against YKL40 (ab77528, 1/1000), sortilin (ab16640, 1/1000), CD9 (ab92726, 1/1000) and HSP90 (ab13492, 1/1000), TSG101 (ab30871, 1/1000) were obtained from Abcam. Antibodies against TrkA (AF175, 1/200) TrkB (MAB397, 1/200), TrkC(AF1404, 1/200) and P75^NTR (AF1157, 1/1000) were purchased from R&D System. Antibodies against NT3 (5237SC, 1/1000), Sox2 (3579S, 1/1000), Oct4 (2750S, 1/1000) and flotillin (18634S, 1/1000) were purchased from Cell Signaling Technology. Antibodies against NGF (sc-548, 1/200) and BDNF (sc-20981, 1/200) were purchased from Santa Cruz Biotechnology.

Pinet S., Bessette B., Vedrenne N., Lacroix A., Richard L., Jauberteau M.O., Battu S, & Lalloué F. (2016). TrkB-containing exosomes promote the transfer of glioblastoma aggressiveness to YKL-40-inactivated glioblastoma cells. Oncotarget, 7(31), 50349-50364.

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Western Blot Analysis of Key Proteins

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Equal amounts of proteins were separated by SDS-PAGE. After blocking the membrane with 5 % skim milk for 1 h at room temperature, the membranes were incubated with primary antibodies against NFAT (sc-271127), MyoD (sc-760), p-MARCKS (sc-101730), and p27 (sc-528; all Santa Cruz Biotechnology), LaminB2 (sab2702205; Thermo Fisher Scientific), and β-actin (A5316; Sigma-Aldrich Corp.) overnight at 4 °C. The membrane was washed and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies. The bands were detected by an enhanced chemiluminescence (ECL) reagent (Santa Cruz Biotechnology). The band intensities were quantified using NIH Image J version 1.34e software.

Seo H.H., Lee C.Y., Lee J., Lim S., Choi E., Park J.C., Lee S, & Hwang K.C. (2016). The role of nuclear factor of activated T cells during phorbol myristate acetate-induced cardiac differentiation of mesenchymal stem cells. Stem Cell Research & Therapy, 7, 90.

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Western Blot Analysis of Cell Signaling

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Protein was collected from the cells after various treatments. For Western blots, a previously described procedure was applied [20 (link)]. The immunoblotting was performed with primary antibodies recognizing p⁴⁷³Ser-Akt (#9271), Akt (#9272), PARP (#9542), cleaved caspase-9 (#7237), p¹⁸⁰Thr/¹⁸²Tyr-p38 MAPK (#9215), p38 MAPK (#9212), HIF-1α (#3716), Mcl-1 (#39224), survivin (#2802), LC3B(#2775), p¹⁵Ser-p53 (#9284), and p53 (#2527) all from Cell Signaling Technologies (Beverly, MA, USA); procaspase-8 (MAB4708) from Millipore; Bax (ab7977) from Abcam (Cambridge, UK); Bcl-2 (Sc-509) from Santa Cruz Biotechnology (CA, USA); Atg5 (GTX62601) from GeneTex (Irvine, CA, USA); β-actin (A5316) from Sigma-Aldrich (St. Louis, MO, USA). Immunoblotted bands were visualized by an enhanced chemiluminescence reagent (GE Healthcare Bioscience, NJ, USA) and system (FUSION SoLo S, Deutschland, Germany) using secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Lin C.W., Bai L.Y., Su J.H., Chiu C.F., Lin W.Y., Huang W.T., Shih M.C., Huang Y.T., Hu J.L, & Weng J.R. (2020). Ilimaquinone Induces Apoptosis and Autophagy in Human Oral Squamous Cell Carcinoma Cells. Biomedicines, 8(9), 296.

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Western Blot Analysis of Cell Lysates

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Cells were lysed with NP-40 cell lysis buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 5 mM MgCl₂, 1 mM EDTA, 10% Glycerol, 1% NP-40), with freshly added Protease inhibitor (11697498001, Roche, Mannheim, Germany) according to the manufacturer's protocol. 20 μg of cell lysate was loaded into each well for SDS-polyacrylamide gel electrophoresis (PAGE) and was electroblotted onto nitrocellulose membranes (10600008, GE healthcare, Freiburg, Germany). Membranes were incubated with antibodies against β-Actin (A5316) from Sigma (Saint Louis, USA); c-myc (SC40) from Santa Cruz (Dallas, USA); ERα (SC543) from Santa Cruz (Dallas, USA); Flag (F3165) from Sigma (Saint Louis, USA); GFP (MAB2510) from Millipore (Temecula, USA); ubiquitin (FL76) from Santa Cruz (Dallas, USA); PAK4 (total) pAb (6508) generated in our laboratory [25 (link)].

Zhuang T., Zhu J., Li Z., Lorent J., Zhao C., Dahlman-Wright K, & Strömblad S. (2015). p21-activated kinase group II small compound inhibitor GNE-2861 perturbs estrogen receptor alpha signaling and restores tamoxifen-sensitivity in breast cancer cells. Oncotarget, 6(41), 43853-43868.

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Protein Expression Analysis by Western Blot

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To assess protein expression, proteins isolated from each patient's wound section were fractionated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. Blots were then probed with respective primary antibodies specific for each protein: β-actin (A5316 from Sigma-Aldrich; NHE1, MCT-1 and ATP6V1B1/B2 from Santa Cruz Biotechnology, CA, USA). Dilutions were made according to the manufacturer's recommendation. After incubation with respective primary antibodies, the membranes were washed in tris-buffered saline supplemented with 0.1% Tween 20 and incubated with the appropriate secondary antibodies (Sigma-Aldrich) followed by development with the BCIP/NBT phosphatase substrate system (Pierce Biotechnology, Rockford, IL). β-actin served as loading control.

Schreml S., Meier R.J., Kirschbaum M., Kong S.C., Gehmert S., Felthaus O., Küchler S., Sharpe J.R., Wöltje K., Weiß K.T., Albert M., Seidl U., Schröder J., Morsczeck C., Prantl L., Duschl C., Pedersen S.F., Gosau M., Berneburg M., Wolfbeis O.S., Landthaler M, & Babilas P. (2014). Luminescent Dual Sensors Reveal Extracellular pH-Gradients and Hypoxia on Chronic Wounds That Disrupt Epidermal Repair. Theranostics, 4(7), 721-735.

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Western Blot Analysis of Cell Signaling Proteins

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Following the drug treatment, cells were scraped in RIPA buffer (Santa Cruz Biotechnology) and the whole cell lysates were sonicated. Samples were run at 6-15 % SDS-PAGE and transferred to 0.22µm nitrocellulose (Bio-Rad) or 0.45μm PVDF (Millipore) membranes. Membranes were probed for the indicated target proteins using primary and secondary antibodies as described before 34 (link). 4E-BP1 (#9452), Bax (#5023), Bcl-2 (#2870), Bcl-xL (#2764), Bim (#2933), caspase 9 (#9502), CDK2 (# 2546), CDK4 (#12790), cleaved caspase 3 (#9664S), Cyclin D3 (#2936), Cyclin E1 (#4129S), Cyclin D1 (#2978S), Mcl-1 (#5453), mTOR (#2972), Noxa (#14766), p53 (#2524), Puma (#4976), p27Kip1 (#3686), p70S6K (#9202), pan-actin (#8456), PARP (#9542), p-4E-BP1 (#9455), p-mTOR (#5536), p-p70S6K (#9205), p-S6 (#4858 and #2215), and S6 (#2317) were purchased from Cell Signaling Technology, β-Actin (A5316) from Sigma Aldrich. The signal was detected by using Chemiluminescent detection kit (Advansta) and visualized by ChemiDoc XRS+ (Bio-Rad). Adobe Photoshop CC 2014 was used to process the images. One representative blot of at least three independent experiments was shown in figures.

Nayman A.H., Siginc H., Zemheri E., Yencilek F., Yildirim A, & Telci D. (2019). Dual-Inhibition of mTOR and Bcl-2 Enhances the Anti-tumor Effect of Everolimus against Renal Cell Carcinoma In Vitro and In Vivo. Journal of Cancer, 10(6), 1466-1478.

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Immunoblotting Antibody Inventory

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Antibodies against AIF-FL (#4642), β-catenin (#9587), Akt (#9272), pAkt (#4060), GSK-3β (#9315), pGSK-3β (#9331), PTEN (#9559), Cox-IV (#4850), and His (#2366) were purchased from Cell Signaling Technology (Danvers, MA); the antibody for AIFsh2 and AIFsh3 (LS-C147989) from Lifespan Biosciences (Seattle, WA); Streptavidin–HRP Conjugate (89880D) from Thermo Fisher Scientific (Waltham, MA); Ndufs1 (12444-1-AP) and Ndufs3 (15066-1-AP) from Proteintech Group (Chicago IL); HA (sc-805) and LaminB (sc-6216) from Santa Cruz; GST (013-21851) from Wako; Flag (A8592) and β-actin (A5316) from Sigma-Aldrich.

Shen S.M., Guo M., Xiong Z., Yu Y., Zhao X.Y., Zhang F.F, & Chen G.Q. (2015). AIF inhibits tumor metastasis by protecting PTEN from oxidation. EMBO Reports, 16(11), 1563-1580.

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Molecular Signaling Pathway Analysis

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Reagents included human GM-CSF (R&D Systems, Minneapoilis, MN), human M-CSF (Chiron, Emeryville, CA), dexamethasone (Dex), and mifepristone (Sigma-Aldrich, St. Louis, MO). Antibodies for Western blotting were against phospho-p38 (D3F9), p38 (D13E1), phospho-JNK1/2 (81E11), JNK1/2 (9252), phospho-ERK1/2 (D13.14.4E), ERK1/2 (137F5), caspase-3 (9662), caspase-8 (D35G2), Bcl2 (50E3), Bcl-xL (54H6), Bid (2002), Bak (D4E4), Bax (D2E11), Bim (C34C5), phospho-Bad S112 (40A9), Bad (D24A9), phospho-RSK S380 (D3H11) and RSK (32D7) (Cell Signaling Technologies, Danvers, MA), and β-actin (A5316) (Sigma-Aldrich).

Achuthan A., Aslam A.S., Nguyen Q., Lam P.Y., Fleetwood A.J., Frye A.T., Louis C., Lee M.C., Smith J.E., Cook A.D., Olshansky M., Turner S.J, & Hamilton J.A. (2018). Glucocorticoids promote apoptosis of proinflammatory monocytes by inhibiting ERK activity. Cell Death & Disease, 9(3), 267.

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