Total protein was extracted using RIPA lysis buffer (1:1000) (Solarbio Science and Technology Co., Ltd., Beijing, China) and protease/phosphate inhibitors (Solarbio Science and Technology Co., Ltd., Beijing, China). The protein concentrations were measured using a bicinchoninic acid protein quantification kit (Solarbio Science and Technology Co., Ltd., Beijing, China). Proteins were separated by 10% or 12.5% SDS-PAGE gel electrophoresis, transferred to PVDF membranes, and probed with primary antibodies TBC1D1 (diluted at 1:1000, Proteintech, China), N-Cadherin, E-Cadherin (diluted at 1:1000, CST, USA), MMP-2, MMP-9 (diluted at 1:1000), Profilin (diluted at 1:10000), Cofilin, Cofilin (phosphoS3) (diluted at 1:1000, Abcam, UK), and Phospho-LIMK1/LIMK2 (Thr508, Thr505) (diluted at 1:1000, Invitrogen). The membranes were probed with horseradish peroxidase-conjugated secondary antibodies. An enhanced chemiluminescence detection system (Bio-Rad, Hercules, CA, USA) was used for protein detection. Anti-GAPDH antibody and anti-β-TUBULIN (diluted at 1:2000, Proteintech) were used to monitor the loading amount.
Anti β tubulin
Manufactured by Proteintech
Sourced in United States, China
Anti-β-tubulin is a primary antibody that specifically recognizes the β-tubulin protein, a major component of the cytoskeleton in eukaryotic cells. This antibody can be used in various immunodetection techniques, such as Western blotting and immunocytochemistry, to study the distribution and expression levels of β-tubulin in biological samples.
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Lab products found in correlation
Anti gapdh, by Proteintech (7 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Anti cse, by Proteintech (3 mentions)
Anti β actin, by Proteintech (3 mentions)
Ripa lysis buffer, by Solarbio (2 mentions)
Anti e cadherin, by Cell Signaling Technology (2 mentions)
Anti lamin b1, by Proteintech (2 mentions)
Ripa lysis buffer, by Sangon (2 mentions)
Anti connexin 43, by Proteintech (2 mentions)
Anti vegfa, by Proteintech (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Bca protein assay kit, by CoWin Biotech (2 mentions)
Rabbit monoclonal anti phospho stat1, by Cell Signaling Technology (2 mentions)
Rabbit monoclonal anti irf1, by Proteintech (2 mentions)
Mouse monoclonal anti lpcat3, by Proteintech (2 mentions)
Anti p eif2α ser51, by Cell Signaling Technology (2 mentions)
Anti chop, by Proteintech (2 mentions)
Anti ubiquitin, by Proteintech (2 mentions)
Anti perk, by Proteintech (2 mentions)
40 protocols using anti β tubulin
1
Protein Extraction and Western Blot Analysis
2
Protein Expression Analysis by Western Blot
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Cells were harvested after the indicated treatments and dissolve RIPA buffer; protein concentrations quantification by bicinchoninic acid detection kit (Pierce, USA). Protein samples were Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separation under denaturing conditions and transferred to a nitrocellulose filter (NC) membrane. The membrane was then incubated with the following primary antibodies: anti-β-tubulin (CST), anti-GAPDH (CST), anti-CDC20 (Proteintech), anti-BCL-2 (CST), anti-BAX (CST), anti-p21 (Proteintech), anti-P53 (Proteintech), anti-CCNB1 (CST), anti-CCND1 (CST).
3
Western Blotting and Immunofluorescence Staining
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For western blotting assays, whole‐cell extracts were denatured, separated on SDS/PAGE gels and transferred to poly(vinylidene difluoride) membranes. After blocking in 5% (w/v) non‐fat dry milk in Tris‐buffered saline with Tween, membranes were probed with primary antibodies overnight at 4 °C. For immunofluorescence staining, cells were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature, permeabilized with 0.5% triton x‐100 in PBS for 5 min, blocked for 1 h at room temperature with 10% normal goat serum in PBS and incubated overnight at 4 °C with primary antibodies. Primary antibodies used were: anti‐β‐catenin (catalog. no. 51067‐2‐AP; ProteinTech, Rosemont, IL, USA), anti‐fibronectin (catalog. no. 15613‐1‐AP; ProteinTech) and anti‐β‐tubulin (catalog. no. 66240‐1‐Ig; ProteinTech).
4
Western Blot Analysis of Jejunal Mucosa
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We used the jejunal mucosa to prepare the protein samples for western blot assay, as previously described [28 (link)–30 (link)]. The jejunal segments samples were collected from the approximately middle positions in the jejunal tracts, as previously described [28 (link), 31 (link)]. The mid-jejunal segments were rinsed with PBS solution, and then the jejunal mucosa samples were gently scraped off. WCLs of the jejunal mucosa were prepared in RIPA lysis buffer (Sangon Biotech, C500005). The nuclear and cytoplasmic extraction of the intestinal epithelium was conducted using a kit (Thermo Fisher Scientific, 78833). This western blot assay was conducted as previously described [31 (link)]. Primary antibodies included the anti-E-cadherin (Cell Signaling Technology, 14472S), anti-ZO-1 (ABclonal, A11417), anti-Connexin 43 (Proteintech, 26980-1-AP), anti-Lamin B1 (Proteintech, 12987-1-AP), anti-CYP1A1 (Proteintech, 13241-1-AP), anti-AhR (Proteintech, 67785-1-Ig), anti-β-actin (Sigma, A5441), and anti-β-tubulin (Proteintech, 66240-1-Ig). Secondary antibodies included the HRP-conjugated secondary antibodies (Cell Signaling Technology, 7076S and 7074S).
5
Separation and Detection of Tubulin Isoforms
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Proteins were separated using 10% gel with an electrophoresis cells (GE Life Technologies). To separate α- and β-tubulins, 10% gel was prepared using a stock solution of 40% acrylamide solution (Meryer) supplemented with 0.54% bisacrylamide (w/v) powder (Yuanye) according to a protocol reported previously (39 (link)). After electrophoresis, proteins were transferred onto a nitrocellulose membrane using a semidry blotter (GE Healthcare). Membranes were incubated with anti-His tag (1:5000 dilution; Immuno Way), anti-glutamylation (GT335, 1:4000 dilution; Adipogen), anti-polyglutamylation (polyE, 1:4000 dilution; Adipogen), anti-α-tubulin (EP1332Y, 1:4000 dilution; Abcam or DM1A, 1:4000 dilution; Sigma), or anti-β-tubulin (anti-β-tubulin, 1:50,000 dilution; Proteintech) antibody. Immunoreactivity of proteins was visualized with WesternBright ECL (Advansta) using Western Blot Imager (Vilber) after incubation with horseradish peroxidase–labeled goat antimouse (1:5000 dilution, Bioss) or donkey anti-rabbit (1:5000 dilution, Bioss) antisera.
6
Caspase-8 expression analysis
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Protein was extracted on ice from embryos at E10.5 using RIPA buffer. Protein concentrations were determined using the Bradford assay. Western blot was performed using conventional methods, with 50-μg protein run per sample on NuPAGE 4–12% Bis-Tris gel (Life technologies), followed by transfer to PVDF membranes (XCell II Blot Module, Invitrogen). The primary antibodies were rabbit anti-caspase-8 (1:500, Proteintech, Chicago, USA) and anti-β-TUBULIN (1:10,000, Proteintech, Chicago, USA). After incubation with secondary antibody (1:10,000, DAKO), blots were developed using ECL Prime (GE Healthcare Life Sciences) or ECL Western Blotting Substrate (Promega). ImageJ was used to detect densitometry. The results were normalized using β-TUBULIN loading control. Independent experiments were carried out three to four times per sample.
7
Anti-inflammatory and Apoptosis Modulation
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Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (USA). BRB, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), HAuCl4. 3H2O (≥99.9% trace metals basis), BSA, Triton X-100 and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (USA). RIPA lysis buffer, phenylmethanesulfonyl fluoride, hematoxylin-eosin (HE) staining kit and Nissl staining solution (methylene blue) were obtained from Solarbio (China). The antibodies to anti-CD86, anti-CD206, anti-Cleaved Caspase-3 and anti-TNF-α were purchased from Cell Signaling Technology (USA). Anti-Bax, anti-Bcl-2, anti-IL-1β and anti-IL-6 were purchased from Affinity (USA). Anti-β-Tubulin, anti-GAPDH, HRP Affinipure Goat Anti-Mouse IgG and HRP Affinipure Goat Anti-Rabbit IgG were purchased from Proteintech (USA). The Alexa Fluor®568 goat anti-mouse/rabbit IgG and Alexa Fluor®488 goat anti-mouse/rabbit IgG were purchased from Invitrogen (USA). 4,6-dimethyl-2-phenylindole (DAPI) was purchased from Abcam (UK). RAW 264.7 cells and VSC 4.1 cells were obtained from the American type culture collection.
8
Quantitative Western Blot Analysis
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Total cellular protein was extracted, and its concentration determined, using the Protein Extraction kit and the BCA protein concentration kit (both Beyotime Institute of Biotechnology), respectively. Protein samples (40 µg) were separated using 10% SDS-PAGE and transferred to a PVDF membrane. After blocking with 5% skim milk for 1 h at room temperature, the membrane was incubated overnight at 4°C with anti-hnRNPAB (cat. no. 14813-1-AP; 1:2,000), anti-Wnt3a (cat. no. 26744-1-AP; 1:2,000), anti-Wnt5a (cat. no. 55184-1-AP; 1:2,000), anti-β-catenin (cat. no. 17565-1-AP; 1:2,000) and anti-β-tubulin (cat. no. 10094-1-AP; 1:5,000) (all ProteinTech Group, Inc.). The following day, the membrane was washed three times with PBS containing 0.1% Tween-20, and incubated with a horseradish peroxidase-conjugated antibody solution (cat. no. SA00001-2; 1:10,000; ProteinTech Group, Inc.) for 1 h at room temperature. Protein bands were visualized using Super ECL plus super-sensitive luminescent solution (Bio-Rad Laboratories, Inc.) and exposed using X-ray film. Quantity One software v4.6.6 (Bio-Rad Laboratories, Inc.) was used to quantify band intensities.
9
Protein Extraction and Western Blot Analysis
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Total protein was extracted from PSCs and lysed in RIPA lysis buffer with 1% PMSF. The protein concentration was detected with Pierce BCA Protein Assay Reagent (Thermo-Fisher, Waltham, MA, USA). The primary antibodies used were anti-DLST (1:1000, Abclonal, Wuhan, China, Cat#A13297), anti-MYOG (1:1000, Abclonal, Wuhan, China, Cat#A17427), anti-MYOD (1:1000, Proteintech, Wuhan, China, Cat#18943–1-AP), anti-myosin heavy chain (MYHC; 1:3000, Millipore, Darmstadt, Germany, Cat#05–716), and anti-β-tubulin (1:3000, Proteintech, Wuhan, China, Cat#10068–1-AP). The HRP conjugated secondary antibodies (1:4000) HRP-labelled goat anti-mouse IgG (Servicebio, Wuhan, China, Cat#GB23301) and HRP-labelled goat anti-rabbit IgG (Servicebio, Wuhan, China, Cat#GB23303) were used as secondary antibodies.
10
Immunoblotting Analysis of Protein Expression
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The cultured cells and cardiac tissues were solubilized in the presence of protease inhibitors as previously described [19 (link)]. After quantification using a BCA Protein Assay Kit (Solarbio), the proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Pall Corporation, Pensacola, FL). The membranes were incubated with the appropriate primary antibodies; the primary antibodies were then detected using peroxidase-conjugated secondary antibodies (1:5000), which were visualized with an enhanced chemiluminescence reagent (ECL, GE Healthcare, Amersham, UK). Immunoblotting was performed with the following antibodies: anti-CSE (1:1000, Proteintech Group, USA), anti-CD36 (1:1000, Proteintech Group, USA), anti-VAMP3 (1:1000, Proteintech Group, USA), anti-Hrd1 (1:1000, Proteintech Group, USA), anti-β-tubulin (1:1000, Proteintech Group, USA), and anti-caveolin (1:1000, Proteintech Group, USA). Densitometry was conducted through image processing, and the data were analyzed with AlphaView SA.
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