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1 408 protocols using β actin

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Western Blot Analysis of Signaling Proteins

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Protein lysate (400 μL) was added to the tissue sample and lysed for 30 min (on ice), and then centrifuged at 10,000 g at 4 °C for 10 min. The protein concentration in the supernatant was determined by using BCA quantitative kit following the manufacturer's instructions (Biyuntian Biology Science and Technology Co., Ltd, China). Protein was electro-transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated with primary antibodies NF-κB (Beijing Bioss biology technology Co., Ltd, China, 1:1000), STAT3 (Beijing Bioss biology technology Co., Ltd, China, 1:1000), JAK2 (Beijing Solarbio Science and Technology Co., Ltd, China, 1:1000), and β-actin (Proteintech Group, US, 1:1000) at 4 °C overnight. Then the membrane was washed three times using TBST and incubated with secondary antibodies HRP-Rabbit-anti-goat (Proteintech Group, US, 1:2000) for 1 h. β-actin served as an internal reference.
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Protein Expression Profiling in Porcine Intestine

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Jejunum and ileum were extracted with total protein extraction reagents (Thermo Fisher Scientific Inc., New York, NK, USA) in accordance with the manufacturer’s instructions. The relative expression of protein was determined by Western blot technique as described previously [22 (link)]. The following antibodies were used for protein quantification: PXR (1:500; Proteintech Group, Inc.), RXRα (1:500; Proteintech Group, Inc.), CYP2B6 (1:200; Proteintech Group, Inc.), CYP3A4 (1:500; Proteintech Group, Inc.), CYP3A5 (1:2000; Proteintech Group, Inc.), NF-κBp65 (1:1000; Cell Signaling Technology, Danvers, MA, USA), phosphorylated NF-κBp65 (Ser536) (1:1000; Cell Signaling Technology, Danvers, MA, USA), IKKα(1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), IκB (1:1500; Proteintech Group, Inc.), IL-10(1:1000; Abcam, Cambridge, LON, UK) and β-actin (1:4000;Proteintech Group, Inc.)and secondary antibody horseradish peroxidase-conjugated goat anti-rabbit IgG (1:6000; Proteintech Group, Inc.) or anti-mouse IgG (1:4000; Proteintech Group, Inc.). All protein measurements were normalized to β-actin (1:4000; Proteintech Group, Inc.) and data are expressed relative to the values in control piglets. In addition, porcine protein CYP2B22, CYP3A29, CYP3A46 have a high homology similarity (over 90 percent) with human protein CYP2B6, CYP3A4, CYP3A5, respectively.
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Western Blot Analysis of Glioblastoma Cell Lines

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Post-transfection, U87 or U251 cells were lysed with buffer containing RIPA for 30 min on ice and then centrifuged at 12,000 g/min for 20 min at 4°C. Cell lysates were assessed for total protein concentration using the BCA protein assay kit. Subsequently, 20 μg protein samples were separated on 10% SDS-PAGE using the Bio-Rad Bis-Tris Gel System (Bio-Rad, CA, USA) and transferred to polyvinylidene fluoride membranes (Millipore, Danvers, MA, USA). Membranes were blocked with 5% non-fat milk for 60 min at room temperature. The primary antibodies used were VEGFA (1:500, Proteintech, USA), VEGF2 (1:500, Proteintech, USA), Akt (1:5000, Proteintech, USA), mTOR(1:5000, Proteintech, USA), and β-actin (1:5000, Proteintech, USA); the corresponding secondary antibodies were anti-mouse and anti-rabbit (Proteintech, USA). Primary antibodies against VEGFA were purchased from Bioss Co., Ltd. (Beijing, China), and primary antibodies against VEGFA, Akt, mTOR, and β-actin were purchased from Proteintech, USA. Finally, proteins were detected by enhanced chemiluminescence (ECL).
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Psoralen Modulation of Wnt/β-catenin Signaling

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To further explore the effects of psoralen on the transcriptional activation of Wnt/β-catenin signaling pathway, western blot analysis was conducted according to the previous protocol20 (link). Using β-actin as an internal reference, the membrane was subsequently incubated at 4 °C overnight with primary antibodies against β-catenin, phospho-β-catenin and Fra-1 diluted at 1:3000, β-actin diluted at 1:5000 (Proteintech Group, CHI, USA), and enhanced chemiluminescence (ECL) plus kit (Millipore, America) was applied for visualization. The β-catenin expression and intracellular localization were analyzed using immunofluorescence. MDA-MB-231 cells and MCF-7 cells were fixed and stained with β-catenin antibodies (1:200 dilution). Images were captured by fluorescent microscopy.
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Quantitative Western Blot Analysis

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Total protein extraction was performed using RIPA lysis buffer (Beyotime), with 1% phenylmethanesulfonyl fluoride (PMSF, Beyotime). An enhanced BCA Protein Assay Kit (Beyotime) was used to prepare a standard curve to measure protein concentrations. SDS-PAGE was performed after loading 15 μl protein at 15-30 μg. Once samples were separated, they were transferred to a PVDF membrane (Invitrogen, Carlsbad, CA, USA). The membrane was blocked in 5% albumin bovine (BSA, Biosharp), followed by incubation with diluted primary antibodies at 4°C overnight: HMGB1 (1:1000, Cell Signaling Technology, Danvers, MA, USA), β-actin (1:2000, Proteintech, Wuhan, China). After washing steps, secondary antibodies were added and incubated at 37°C for 40 min: goat anti-mouse IgG-HRP (1:10000, Proteintech) for β-actin, and goat anti-rabbit IgG-HRP (1:10000, Proteintech) for HMGB1. Enhanced chemiluminescence (ECL) reagent (7-Sea Biotech, Shanghai, China) was added to membranes to delineate protein signals, after which membranes were scanned and analyzed using Image acquisition and analysis system (WD-9413B, Liuyi, Beijing, China) and Gel-Pro-Analyzer software (Media Cybernetics, USA).
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Quantification of Mitochondrial Dynamics Proteins

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BCA Protein Colorimetric Assay Kit (Elabscience Biotechnology Co., Ltd., China) was used to quantify the total protein of brain cortex tissues and SY5Y cells. Samples with an equal amount of protein were loaded and separated via sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The proteins were transferred to the appropriate polyvinylidene fluoride membrane (Millipore, USA), blocked with 5% skimmed milk for 1 h, and thens incubated with diluted antibodies at 4°C overnight. For the in vivo experiments, Drp1 (1:2000), Fis1 (1:1500), MFF (1:8000), OPA1 (1:2000), Mfn1 (1:1000), Mfn2 (1:2000), and β‐actin (1:5000) antibodies were purchased from Proteintech Group, Inc., China. Meanwhile, for the in vitro experiments, the antibodies included Drp1 (1:800; AiFang Biological, China), Fis1 (1:1500; Bioss, China), OPA1 (1:1000; AiFang Biological, China), Mfn1 (1:1000; Affinity, China), Mfn2 (1:500; AiFang Biological, China), and SENP6 (1:1000; Abcam, USA). Furthermore, MFF (1:2000), SUMO1 (1:2000), SUMO2/3 (1:500), SENP1 (1:2000), SENP2 (1:1000), SENP3 (1:800), SENP5 (1:3000), and β‐actin (1:5000) were obtained from Proteintech Group, Inc., China. Then, the membranes were washed and incubated with secondary antibodies for 2 h at room temperature. After rewashing with TBST buffer, the membranes were added ECL reagent to enable the detection of the protein bands.
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Western Blot Analysis of Protein Expressions

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U87 or U251 cells 6 h after transfection were lysed by lysis buffer with RIPA on ice for 30 min and centrifuge at 4°C for 20 min at 12000g/min. The total protein concentration of cell lysates was assessed using the BCA Protein Assay kit. Subsequently, 20 μg protein samples were separated on a 10% SDS-PAGE by the use of a Bio-Rad Bis-Tris Gel system (Bio-Rad, CA, USA), transferred onto polyvinylidene difluoride membranes (Millipore, Danvers, MA, USA), blocked with 5% nonfat milk for 60 min at room temperature. Primary antibodies were WEE1 (1 : 1000, Proteintech, USA) and β-actin (1 : 5000, Proteintech, USA); corresponding secondary antibodies were anti-mouse and anti-rabbit (Proteintech, USA). Primary antibodies against WEE1 were purchased from Bioss Co., Ltd. (Beijing, China) and the primary antibodies against N-cadherin, vimentin, and β-actin (Proteintech, USA). Finally, the proteins were detected by enhanced chemiluminescence (ECL) and then exposed about 60 s for scanning and measuring.
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GLUT1 Protein Expression Analysis

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After treatment of RIPA buffer (Solarbio) with 1% protease inhibitor, total protein was extracted after centrifugation. The protein concentration was detected via a BCA kit (Sigma, St. Louis, MO, USA). The protein samples (20 μg) were separated via SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Solarbio). Following blocking in 5% non-fat milk, the membranes were interacted with antibody for GLUT1 (Cat. No. 21829-1-AP, 1:1000 dilution, Proteintech, Rosemont, IL, USA) or β-actin (Cat. No. 20536-1-AP, 1:3000 dilution, Proteintech) overnight and IgG conjugated via HRP (Cat. No. SA00001-2, 1:10,000 dilution, Proteintech) for 2 h. β-actin was employed as a reference. The blots were visualized by ECL reagent (Thermo Fisher), and then analyzed via Image J software (NIH, Bethesda, MD, USA). The relative protein expression was normalized to the control group. Each sample was provided in quadruplicate, and this experiment was performed 3 times.
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Dendrobium officinale Immune Regulation Protocol

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Dendrobium officinale was purchased from Zhejiang Shouxiangu Pharmaceutical Co., Ltd. (Jinhua, China), and PMA and LPS were purchased from Sigma (Saint Louis, MI, USA). Recombinant human interleukin-4 (IL-4) was purchased from PeproTech, Inc. (Rocky Hill, NJ, USA). TNF-α and IL-6 ELISA kits were purchased from Lianke Biological Technology (Hangzhou, China). Cell Counting Kit-8 (CCK-8) Assay Kit was received from MCE (Romulus, NJ, USA). Total Protein Extraction kit was purchased from Beyotime (Shanghai, China). The MiNiBEST Universal RNA Extraction Kit, PrimeScript RT Reagent kit, and SYBR Premix Ex Taq II Kit were purchased from TaKaRa (Dalian, China). Transwell polycarbonate membrane had an 8 μm pore size (Corning City, NY, USA). The primary antibodies E-cadherin, N-cadherin, Vimentin, Caspase-3, Bax, Bcl-2, Ki67, and β-actin were purchased from Proteintech (Wuhan, China). The primary antibodies ARG1, TGM2, and Cleaved-NOTCH1 were purchased from Cell Signaling Technology (Danvers, MA, USA). The primary antibodies STAT6, p-STAT6, PPAR-r, JAGGED1, Cleaved-NOTCH1, and NOTCH1 were purchased from Abcam (Cambridge, UK). β-actin was purchased from Proteintech (Wuhan, China). The ECL Plus Western Blotting Detection Kit was purchased from Technology Co., Ltd. (Beijing, China). Anti-CD80-FITC and Anti-CD206-PE were obtained from Thermo Fisher (Waltham, MA, USA).
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Mechanism of BDE-47-induced Oxidative Stress

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BDE-47 (≥99.9%) was obtained from CSNpharm (Chicago, IL, USA). ISL (≥98%, HPLC) and LCB (≥98%, HPLC) were obtained from Yuanye (Shanghai, China). DCFH-DA and DMSO were obtained from Solarbio (Beijing, China). An apoptosis kit was obtained from Multi science (Hangzhou, China). A DAPI dye and neutral red staining solution were obtained from Beyotime Biotechnology (Shanghai, China). ELISA kits were obtained from Mlbio (Shanghai, China) (TNF-α Cat. No. ml002095; IL-6 Cat. No. ml063159; IL-1β Cat. No. ml301814). CAT, SOD, and GSH kits were obtained from Solarbio (Beijing, China). β-Actin, Nrf2, Keap1, HO-1, NQO1, IKBKB, IκB-Alpha, Bax, Bcl-2 antibodies, and the ECL luminescent reagent were obtained from Proteintech (Wuhan, China) (β-Actin Cat. No. 66009-1-lg; Nrf2 Cat. No. 16396-1-AP; Keap1 Cat. No. 60027-1-lg; HO-1 Cat. No. 66743-1-lg; NQO1 Cat. No. 67240-1-lg; IKBKB Cat. No. 15649-1-AP; IκB-Alpha Cat. No. 10268-1-AP; Bax Cat. No. 50599-2-lg; Bcl-2 Cat. No. 26593-1-AP). Caspase-3 antibody was obtained from BBI (Shanghai, China) (Cat. No. D16009). NF-κB and p-NF-κB antibodies were obtained from Abcam (Cambridge, UK) (NF-κB Cat. No. ab76302; p-NF-κB Cat. No. ab32536). P-Nrf2 antibody was obtained from Abclonal (Wuhan, China) (Cat. No. AP1133). Real-time fluorescent quantitative reagents were obtained from TransGen Biotech (Beijing, China).
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