As outlined in previous publications [30 (link)-32 (link)], HCECs exposed to A. fumigatus for 8 h were lysed in a mixture of RIPA buffer (Solarbio, Beijing, China), phenylmethanesulfonyl fluoride (Solarbio), and phosphatase inhibitor (MCE; mixing ratio: 98:1:1) for 2 h. The protein concentration was quantified by bicinchoninic acid (BCA) assay (Elabscience, Wuhan, China). The protein samples separated by 10% sodium salt -polyacrylamide gel electrophoresis (SDS-PAGE) were transferred onto polyvinylidene difluoride membranes. The membranes blocked with blocking buffer were incubated with anti-β-actin antibody (1:3000; Elabscience) and anti-HO-1 antibody (Abcam;1:10000) at 4 °C overnight. After being washed in PBST and incubated with the corresponding secondary antibodies for 1 h, the bands on the membranes were detected with enhanced chemiluminescence (ECL) reagents (Biorad, CA, America). A quantitative grayscale analysis of the western blot bands was performed using Image J.
Anti β actin antibody
Manufactured by Elabscience
Sourced in United States
The Anti-β-actin antibody is a primary antibody that specifically recognizes the β-actin protein, a ubiquitously expressed cytoskeletal protein that plays a crucial role in various cellular processes. This antibody can be used for the detection and quantification of β-actin in a wide range of applications, including Western blotting, immunohistochemistry, and immunocytochemistry.
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Lab products found in correlation
3 protocols using anti β actin antibody
1
HO-1 Expression in Aspergillus-Exposed HCECs
2
Western Blot Analysis of CTGF and Phospho-Smad1/5
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The cells were lysed with lysis buffer (1% SDS, 10 mm Tris-HCl, pH 7.6, 20 g/ml aprotinin, 20 g/ml leupeptin and 1 mm AEBSF). Protein concentrations were determined using the Bradford method (48 (link)). Protein (20 µg) was separated on 12% SDS-PAGE gels and transferred onto PVDF membranes (Merck Millipore). After blocking with 10% skim milk, the membranes were incubated with the primary antibodies (anti-CTGF antibody, 1:1,000, cat. no. ab6992, Abcam; anti-phospho-Smad1/5 antibody, 1:1,000, cat. no. 9516, Cell Signaling Technology) at 4°C overnight. After washing 3 times with triethanolamine buffer solution (Sangon Biotech Co., Ltd.), the membranes were incubated with goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies (cat. no. 31460; Invitrogen; Thermo Fisher Scientific) (1:200 dilution in 5% skim milk) at room temperature for 1 h. The signals were examined with the ECL kit (Elabscience Biotechnology Co., Ltd.), using anti-β-actin antibody (1:1,000, cat. no. 4970; Cell Signaling Technology) as an internal control. Protein gray value detection was performed using ImageJ software (National Institutes of Health).
3
Hippocampal mTOR Protein Expression
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The hippocampal tissues were dissected quickly on ice and stored at -80°C for later use. Total protein extraction and Western blotting analysis were performed according to previously described methods [46] . Proteins (35 mg per lane) were separated by 8% SDS-PAGE and subsequently transferred to nitrocellulose membranes.
The blots were blocked with 5% skimmed milk powder in 0.1% Tris-buffered saline/Tween-20 at room temperature for 2 h, then incubated overnight at 4°C with primary antibodies for the detection of mTOR (1:2,000, Abcam, USA); anti-β-actin antibody (1:1,000, Elabscience, Wuhan, China) was used as a reference. After three washes with Tris-buffered saline/Tween-20, the membrane was incubated with the horseradish peroxidase-conjugated secondary antibodies (1:200, Zhongshanjinqiao, Beijing, China) for 1 h at room temperature. Immunoreactive proteins were detected and visualized using the enhanced chemiluminescence Western blot detection system (Tanon 5200 Imager, China). To control for sampling errors, the band intensities were normalized against β-actin to quantify the relative protein expression levels.
The blots were blocked with 5% skimmed milk powder in 0.1% Tris-buffered saline/Tween-20 at room temperature for 2 h, then incubated overnight at 4°C with primary antibodies for the detection of mTOR (1:2,000, Abcam, USA); anti-β-actin antibody (1:1,000, Elabscience, Wuhan, China) was used as a reference. After three washes with Tris-buffered saline/Tween-20, the membrane was incubated with the horseradish peroxidase-conjugated secondary antibodies (1:200, Zhongshanjinqiao, Beijing, China) for 1 h at room temperature. Immunoreactive proteins were detected and visualized using the enhanced chemiluminescence Western blot detection system (Tanon 5200 Imager, China). To control for sampling errors, the band intensities were normalized against β-actin to quantify the relative protein expression levels.
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