Anti β actin

Manufactured by Fortis Life Sciences

Sourced in United States

Anti-β-actin is a primary antibody used to detect the presence and quantify the levels of the β-actin protein, a widely expressed cytoskeletal protein, in biological samples. It serves as a reliable loading control and normalization factor in various protein analysis techniques.

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15 protocols using anti β actin

Western Blot Analysis of Bone Markers

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Cell lysates were extracted in 0.1 M NaCl, 0.01 M tris–HCl (pH 7.6), 1 mM EDTA (pH 8.0), 1 mg/ml aprotinin, and 100 mg/ml PMSF; protein concentrations were determined by a Bio-Rad protein assay. Protein (50 μg) was boiled at 95 °C in sodium dodecyl sulphate (SDS) sample buffer for 5 min, electrophoresed on 10% or 12% SDS-PAGE gels, and transferred to polyvinyldifluoridine membranes. Then, blots were incubated overnight at 4 °C with anti-HOXA9 (Abcam, Cambridge, UK, #ab191178; 1:1000), anti-RUNX2 (Abcam, Cambridge, UK, #ab23981; 1:1000), or anti-β-actin (Bethyl, Montgomery, Texas, USA, #A300-491A; 1:8000) antibodies. Membranes incubated with anti-goat or anti-rabbit secondary antibody (Santa Cruz Biotechnology; 1:5000) at room temperature for 60 min. Protein band signals were visualized using an ECF western blotting kit (Amersham Biosciences, Piscataway, NJ, USA) and detected with an LAS3000 luminescent image analyzer (Fuji Photo Film).

Jin Y., Kim H.K., Lee J., Soh E.Y., Kim J.H., Song I., Chung Y.S, & Choi Y.J. (2019). Transcription Factor HOXA9 is Linked to the Calcification and Invasion of Papillary Thyroid Carcinoma. Scientific Reports, 9, 6773.

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Quantitative Immunoblot Analysis of mTOR Signaling

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Equal amounts of protein were analyzed in duplicate by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The following monoclonal antibodies were used: anti–phospho-Akt (Ser473, Cell signaling, Danvers, MA), anti–total Akt (Cell signaling), anti-phospho-mTOR (Ser2448, Cell signaling), anti-phospho-mTOR (Thr2446, Millipore, Bedford, MA), anti-total mTOR (Cell signaling), anti-HIF-1α (Cayman Chemical, Ann Arbor, MI), anti-VHL (Cell signaling), anti-phospho-AMPK (Thr172, Cell signaling), anti-total AMPK (Cell signaling) anti-phospho-P70S6K (Thr389, Cell signaling), anti-total P70S6K (Cell signaling), anti-phospho-4E-BP (Thr37/46, Cell signaling), anti-lactate dehydrogenase A (LDHA) (Cell signaling), anti-hexokinase-2 (HK-2, SantaCruz Biotechnology, Dallas, TX), anti-phospho-TSC2 (Ser1387) (Cell signaling), anti-total TSC2 (Cell signaling), and anti-β-actin (Bethyl, Montgomery, TX, US). Immunoreactive proteins were detected by horseradish peroxidase–conjugated secondary antibodies and enhanced using chemiluminescence (ECL) reagents (GE Healthcare Life Sciences, Piscataway, NJ). All immunoblots were performed with triplicate and visualized by LAS image analyzer (Fujifilm, Tokyo, Japan). The band density was quantified using MultiGauge (Fujifilm).

Woo Y.M., Shin Y., Lee E.J., Lee S., Jeong S.H., Kong H.K., Park E.Y., Kim H.K., Han J., Chang M, & Park J.H. (2015). Inhibition of Aerobic Glycolysis Represses Akt/mTOR/HIF-1α Axis and Restores Tamoxifen Sensitivity in Antiestrogen-Resistant Breast Cancer Cells. PLoS ONE, 10(7), e0132285.

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Protein Extraction and Immunoblotting

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Proteins extracts were prepared using a NucleoSpin® DNA/RNA/Protein kit (MACHEREY-NAGEL), following the manufacturer's protocol. Immunoblotting was carried out according to a previously described method using anti-LY6K (I-14; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-β-actin (Bethyl Laboratories, Inc. Montgomery, TX, USA) antibodies [23 (link)].

Kong H.K., Park S.J., Kim Y.S., Kim K.M., Lee H.W., Kang H.G., Woo Y.M., Park E.Y., Ko J.Y., Suzuki H., Chun K.H., Song E., Jang K.Y, & Park J.H. (2016). Epigenetic activation of LY6K predicts the presence of metastasis and poor prognosis in breast carcinoma. Oncotarget, 7(34), 55677-55689.

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Biochemical Analysis of B Cell Populations

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Wild type follicular B cells and CD11b⁺ATA B lymphoma cells were purified by cell sorting (2 × 10⁶ cells/tube), and cell lysates were subjected to SDS-PAGE, and immunoblotted with anti-IL-10 (R&D Systems), anti-Tyr⁷⁰⁵pStat3 (Abcam), anti-Stat 3 (Santa Cruz), and anti-β actin (Bethyl Lab.).

Hayakawa K., Formica A.M., Nakao Y., Ichikawa D., Shinton S.A., Brill-Dashoff J., Smith M.R., Morse HC I.I.I, & Hardy R.R. (2018). Early Generated B-1–Derived B Cells Have the Capacity To Progress To Become Mantle Cell Lymphoma–like Neoplasia in Aged Mice. The Journal of Immunology Author Choice, 201(2), 804-813.

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Quantitative Analysis of Glucocorticoid Receptor Isoforms

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Cytosolic and nuclear protein fractions (60 μg) were electrophoresed on 3–8% Tris-acetate precast gels (Invitrogen, Life technologies, Carlsbad, California, USA) as previously described [11 (link)]. Blots were incubated with affinity purified polyclonal rabbit anti-human GR total (1:1500) (Bethyl Laboratories, Montgomery, TX, USA, Cat no. A303-491A) antibodies targeted to residue 150–200 of the GR receptor. The appropriate secondary antibody (goat anti-rabbit; 1:2500) was applied for 1 h. Membranes were subsequently probed with anti-β actin (1:4000, Bethyl laboratories, USA Cat no. A300-491A) and anti-lamin A/C (1:1500, Santa Cruz Biotechnology, Santa Cruz, California, USA Cat no. Sc-6215) antibodies as loading controls for cytoplasmic and nuclear fractions, respectively. The densitometric analysis was carried out using G:BOX Chemi Gel Imaging Systems (SYNGENE) to quantify the expression levels of different GR isoforms relative to β actin. Peptide competition with anti GR total antibody (1 μg/1.5 ml) incubated with 1× (1 μg) and 2× (2 μg) concentration of the control peptide (Bethyl Laboratories, USA Cat no: BP303-491A) was performed as a specificity control.

Saif Z., Hodyl N.A., Stark M.J., Fuller P.J., Cole T., Lu N, & Clifton V.L. (2015). Expression of eight glucocorticoid receptor isoforms in the human preterm placenta vary with fetal sex and birthweight. Placenta, 36(7), 723-730.

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Protein Expression Analysis in Cell and Tissue Lysates

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After the experiments were completed, the C2C12 myotubes, mice skeletal muscle, and liver tissue lysates were prepared using a standard protocol. In brief, the cell and tissue samples were mixed with sample buffer (250 mM tris-hydrochloride (pH 6.8), 0.5 M dithiothreitol (DTT), 10% sodium dodecyl sulfate (SDS), 0.5% bromophenol blue, 50% glycerol, and 5% 2-mercaptoethanol) and denatured at 95°C for 5 min. Sample proteins (50 µg) were separated via 10% SDS-polyacrylamide gel electrophoresis (PAGE) and electrotransferred to nitrocellulose transfer membranes (Whatman, 401396, Dassel, Germany), which were incubated overnight at 4°C with 5% skim milk or 5% bovine serum albumin and a range of antibodies. Anti-AMPK, anti-phospho-(p)-AMPK, anti-AKT, anti-p-AKT, anti-GLUT4 (Bioworld Technology), and anti-β-actin (Bethyl Laboratories) antibodies were the primary antibodies used, and a horseradish peroxidase- (HRP-) conjugated goat anti-rabbit immunoglobulin G (IgG) (Santa Cruz Biotechnology) was used as the secondary antibody. The antigen-antibody reaction was detected using a SuperSignal West Femto maximum sensitivity substrate (Thermo Fisher Scientific, Rockford, IL, USA).

Zhao P., Alam M.B., Lee S.H., Kim Y.J., Lee S., An H., Choi H.J., Son H.U., Park C.H., Kim H.H, & Lee S.H. (2017). Spatholobus suberectus Exhibits Antidiabetic Activity In Vitro and In Vivo through Activation of AKT-AMPK Pathway. Evidence-based Complementary and Alternative Medicine : eCAM, 2017, 6091923.

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Western Blot Analysis of Membrane Proteins

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Equal amounts of protein were analyzed in duplicate by SDS-PAGE. The following monoclonal antibodies were used: anti-MUPCDH (anti-μ-protocadherin, C-20; Santa Cruz Biotechnology), anti-AP-2α (Santa Cruz Biotechnology), anti-AQP1 (Santa Cruz Biotechnology), and anti-β-actin (Bethyl Laboratories, Montgomery, TX, USA). Immunoreactive proteins were detected by horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence reagents (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). All immunoblots were performed with triplicate and visualized by LAS image analyzer (Fujifilm, Tokyo, Japan). The band density was quantified using MultiGauge (Fujifilm).

Mi Woo Y., Shin Y., Hwang J.A., Hwang Y.H., Lee S., Young Park E., Kyung Kong H., Cho Park H., Lee Y.S, & Hoon Park J. (2015). Epigenetic silencing of the MUPCDH gene as a possible prognostic biomarker for cyst growth in ADPKD. Scientific Reports, 5, 15238.

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Immunoblotting and Immunoprecipitation Assay

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For immunoblotting, phosphate-buffered saline-resuspended cell lysates were mixed with 2X SDS sample buffer in a 1:1 ratio and boiled for 10 min. For immunoprecipitation, cells were lysed in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 0.5% Triton X-100, and a protease inhibitor cocktail (Roche, Switzerland). Proteins from cell extracts were sequentially immunoprecipitated with anti-Flag M2 agarose (A2220; Sigma-Aldrich) overnight at 4°C and Protein G-Sepharose (GE Healthcare) for 2 h at 4°C. Samples were mixed with the 2X SDS sample buffer in a 1:1 ratio and boiled for 5 min. Immunoprecipitated proteins were analyzed by western blotting using anti-HA (12013819001; Sigma-Aldrich), anti-Flag (F3165; Sigma-Aldrich), and anti-β-actin (A300-491A; Bethyl Laboratories, USA). Proteins were detected using enhanced chemiluminescence reagents (GE Healthcare) according to the manufacturer’s instructions. MG132 was purchased from A.G. Scientific (M-1157).

Lee H.S., Kim M.W., Jin K.S., Shin H.C., Kim W.K., Lee S.C., Kim S.J., Lee E.W, & Ku B. (2021). Molecular Analysis of the Interaction between Human PTPN21 and the Oncoprotein E7 from Human Papillomavirus Genotype 18. Molecules and Cells, 44(1), 26-37.

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Western Blot Analysis of Signaling Proteins

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Western blot analysis was performed as previously described (24 (link)). The following antibodies were used: Anti-phospho-Scr (1:250; rabbit monoclonal; cat. no. 6943S; Cell Signaling Technology, Danvers, MA, USA), anti-Scr (1:1,000; mouse monoclonal; cat. no. 2110s; Cell Signaling Technology), anti-phospho-Cav-1 (1:250; rabbit polyclonal; cat. no. 3251s; Cell Signaling Technology), anti-Cav-1 (1:1,000; rabbit polyclonal; cat. no. sc-894; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-phospho-Akt (1:500; rabbit polyclonal; cat. no. 9271L; Cell Signaling Technology), anti-Akt (1:1,000; rabbit polyclonal; cat. no. 9272S; Cell Signaling Technology), anti-phospho-ERK1/2 (1:500; rabbit polyclonal; cat. no. sc-16982; Santa Cruz Biotechnology), anti-ERK1/2 (1:1,000; rabbit polyclonal; cat. no. 9102S; Santa Cruz Biotechnology), anti-RANK (1:500; rabbit polyclonal; cat. no. A303-897A; Bethyl Laboratories, Inc., Montgomery, TX, USA), anti-β-actin (1:1,000; rabbit polyclonal; cat. no. sc-1616-R; Santa Cruz Biotechnology), followed by incubation with appropriate secondary antibodies. Secondary goat anti-rabbit (1:1,000) and goat anti-mouse antibodies were purchased from Santa Cruz Biotechnology, Inc.

Wang Y., Song Y., Che X., Zhang L., Wang Q., Zhang X., Qu J., Li Z., Xu L., Zhang Y., Fan Y., Hou K., Liu Y, & Qu X. (2018). Caveolin-1 enhances RANKL-induced gastric cancer cell migration. Oncology Reports, 40(3), 1287-1296.

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Purification and Stimulation of B Cell Subsets

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For B cell subset purification for Western blot, FO B cells (CD21^medCD23⁺AA4⁻) and MZ B cells (CD21^hiCD23⁻AA4⁻) from spleen and B1a cells (B220^loCD5⁺) from the PerC were used. To purify μκTg (IgM^a)⁺ FO B and MZ B cells on a C.B17 mouse background, anti–IgM^b antibody was added to exclude endogenous B cells. B1a cells were sorted from both the spleen and PerC of V_H11t.CB17 mice (Wen et al., 2005b (link)). B cell subsets purified by cell sorting (2 × 10⁶ cells per tube) were preincubated in RPMI 1640 plus 0.5% FCS medium in 0.5 ml for 6 h and then stimulated with C12-iE-DAP and/or anti-IgM. Cell lysates were subjected to 10% SDS-PAGE. The Cell signaling analysis reagents used were anti-IκBα, anti–P-IκBα (Ser32), anti-P-p44/p42 MAPK (Erk1/2; Thr202/Tyr204), anti-P-p38 MAPK (Thr180/Tyr182), anti–P-SAPK/JNK (Thr183/Tyr185), anti–P-Akt(Ser473 and Thr308), anti–P-FoxO1 (Ser256), and anti–P-GSK3β (Ser9); protein was detected using HRP anti–rabbit IgG antibody (all from Cell Signaling Technology). The inhibitor LY294002 (Cell Signaling Technology) was used. Anti–β actin (Bethyl Lab), anti-Nod1 (LSBio), and anti-Ptpn22 (Proteintech) Western blots were done without preincubation.

Hayakawa K., Formica A.M., Zhou Y., Ichikawa D., Asano M., Li Y.S., Shinton S.A., Brill-Dashoff J., Núñez G, & Hardy R.R. (2017). NLR Nod1 signaling promotes survival of BCR-engaged mature B cells through up-regulated Nod1 as a positive outcome. The Journal of Experimental Medicine, 214(10), 3067-3083.

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