Colon (proximal) samples were homogenized in mammalian protein extraction reagent (MPER; ThermoFisher Scientific) and centrifuged at 10,000 g for 20 min at 4°C. Expression of candidate proteins in the supernatant were analyzed using western immunoblotting following separation of proteins by SDS-PAGE on a 6 or 12 % gel under denaturing conditions. The membranes were incubated in primary antibody to the following proteins: 15-Pgdh (1:1000, Abcam), Cox-2 (1:500, Abcam), or Zo-1 (1:500, Proteintech) overnight at 4°C. Expression of β-actin (1:20,000; Fitzgerald) or Vinculin (1:5,000; Cell Proteintech) were used as loading controls. The blots were incubated with appropriate HRP-conjugated secondary antibody for 1 hour at room teMPERature and were developed and imaged with West Pico chemiluminescence reagent (ThermoFisher Scientific) and G:BOX Chemi XX6 (Syngene), respectively. Densitometric evaluation of the bands was performed using Image J software (National Institutes of Health).
β actin
Manufactured by Biosynth
Sourced in United States
β-actin is a highly conserved cytoskeletal protein that is essential for cellular structure and function. It is a core component of the microfilament system and plays a crucial role in various cellular processes, including cell motility, cell division, and cell signaling.
Automatically generated - may contain errors
Lab products found in correlation
West pico chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
M per, by Thermo Fisher Scientific (1 mentions)
Cox 2, by Abcam (1 mentions)
15 pgdh, by Abcam (1 mentions)
G box chemi xx6, by Syngene (1 mentions)
Menadione, by Merck Group (1 mentions)
Ab109506, by Abcam (1 mentions)
Txnip antibody, by Proteintech (1 mentions)
Ex 527, by Merck Group (1 mentions)
N ethylmaleimide nem, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
Catalase, by Merck Group (1 mentions)
D alanine, by Merck Group (1 mentions)
Dcp bio1, by Kerafast (1 mentions)
Vimentin, by Biosynth (1 mentions)
Mmp 9, by Cell Signaling Technology (1 mentions)
3 protocols using β actin
1
Quantifying Colon Protein Expression
2
Redox Signaling Pathway Antibody Analysis
3
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Western blot assays were performed to measure the expression of target proteins. Total cellular proteins were extracted from the cultured cells using lysis buffer [20 mM Tris (pH7.4), 150 mM NaCl, 5 mM EDTA, 50 mM NaF and 0.1% NP-40]. Proteins were separated by SDS-PAGE (10%, w/v) and transferred onto PVDF membranes, which were then blocked using 5% skim milk for 2 h at room temperature. The membranes were subsequently incubated with target primary antibodies against the following: MMP-9 [13667S; Cell Signaling Technology (CST), Danvers, MA, USA], vimentin (10R-1903; Fitzgerald Industries International, Acton, MA, USA), α-smooth muscle actin (α-SMA; 19245; CST), TGFBR1 (70R-36484) and β-actin (10R-10263) (both from Fitzgerald Industries International) at 4°C overnight. The dilution used for MMP-9, vimentin, α-SMA and TGFBR1 was 1:100, and the dilution used for β-actin was 1:1,000. Following incubation with horseradish peroxidase-labeled secondary antibodies (A0216; Byotime, Shanghai, China) which were diluted at 1:1,000 at 37°C for 2 h, the protein bands were visualized using a chemiluminescence (ECL) detection system and images were captured.
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