Western blot assay was implemented to examine the protein level of TUBB2A in AGS and HGC-27 cells. Generally, GC cell lysates were harvested with radioimmunoprecipitation buffer (RIPA, Beyotime, Shanghai, China) including protease inhibitor (Beyotime), and quantified by bicinchoninic acid (BCA, Beyotime). Extracted proteins were treated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto nitrocellulose membranes (Millipore Corp, Bedford, MA, USA), followed by blockage with 5% skim milk for 2 h. After incubating with primary antibodies against TUBB2A (1: 1000, ab170931, ABCAm, Cambridge, MA, USA) and β-actin (1: 1000, ab52614, ABCAm)at 4°C all night, membranes were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h. At last, bands were detected by enhanced chemiluminescence reagent (ECL, Pierce Biotechnology, Rockford, Illinois, United States).
Ab52614
Manufactured by Abcam
Sourced in United States
Ab52614 is an antibody reagent developed by Abcam. It is intended for use in laboratory research applications. The core function of this product is to serve as a tool for scientific investigation, but no further details about its intended use can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Merck Group (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Bicinchoninic acid (bca), by Beyotime (1 mentions)
Ab170931, by Abcam (1 mentions)
α sma, by Abcam (1 mentions)
Hmgb1, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
Hydroxyproline hyp assay kit, by Nanjing Jiancheng (1 mentions)
Ab5694, by Abcam (1 mentions)
Ab79823, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Any kd mini protean tgx, by Bio-Rad (1 mentions)
Odyssey clx ir imaging system, by LI COR (1 mentions)
4 protocols using ab52614
1
Quantification of TUBB2A Protein Levels
2
Quantifying Extracellular Matrix Proteins
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The primary antibodies described in this paper including β-actin (ab52614, 1:5000; Abcam, Cambridge, MA, United States), HMGB1 (ab79823, 1:5000; Abcam, Cambridge, MA, United States), α-SMA (ab5694, 1:300; Abcam, Cambridge, MA, United States), goat anti-mouse lgG (ZB-2305, 1:10000; ZSGB-BIO, Beijing, China) and goat anti-rabbit lgG (ZB-2301, 1:10000; ZSGB-BIO, Beijing, China). In addition, the masson trichrome kit (Masson, MST-8003/8004, Maixin-Bio, China), hydroxyproline (HYP) assay kit (A030-2, Nanjing Jiancheng Bioengineering Institute, China), as well as other ELISA kits including HMGB1 (YY42027), type III collagen (Col-III, YY41621), hyaluronic acid (HA, YY42052) and laminin (LN, YY41730) were from Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China) in this study.
3
Protein Extraction and Western Blotting
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Tissue was lysed in denaturing lysis buffer (RIPA) adding dithiothreitol (DTT) to 20 mM before use, followed by SDS-PAGE and immunoblotting. The following antibodies were used for western blotting: rabbit monoclonal caldesmon antibody (Abcam ab45691, Cambridge, MA, USA), rabbit polyclonal IL-10 antibody (Abcam ab9735, Cambridge, MA, USA), and rabbit polyclonal β-actin antibody (Abcam ab52614, Cambridge, MA, USA).
4
Protein Extraction and Western Blot Analysis
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Cells were lysed with M-PER Mammalian Protein Extraction Reagent ThermoFisher (Hemel Hemstead, UK), supplemented with 1× Halt protease inhibitor cocktail ThermoFisher (Hemel Hemstead, UK). Protein concentration was determined using the BCA assay (Pierce). Total protein lysates (20 μg) were separated under denaturing conditions in Any kD Mini-Protean TGX (BioRad, USA) gels. Proteins were then transferred onto polyvinylidene difluoride membranes (BioRad, USA). Membranes were blocked in 1% non-fat milk and incubated overnight at 4°C with anti-β-actin (1:7000 dilution; ab52614, Abcam) and anti-AAG (1:500 dilution; HPA006531, Sigma-Aldrich) antibodies. After primary antibody incubation, membranes were washed and then incubated with the secondary antibodies IRDye 680RD green goat anti-rabbit IgG and IRDye 800CW red goat anti-mouse IgM (LI-COR Biosciences, Lincoln, USA) at 1:10 000 for 1 h at room temperature. Proteins were detected using the Odyssey CLx IR imaging system (LI-COR Biosciences, Lincoln, USA).
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