The tissue samples were homogenized in a tissue grinder. The tissue homogenates were placed on ice, lysed with protein lysis buffer (RIPA: PMSF = 50:1; Beyotime Biotechnology, China) for 30 minutes at 4°C. The lysates were centrifuged for 20 minutes at a speed of 16,000 rpm. The cell lysates were collected into new Eppendorf tubes. Protein concentration was detected by BCA Protein Assay Kit (Coolaber, China). Then, 20 ug of protein were resolved with 10% SDS-PAGE (Sparkjade, China) and electro-transferred to nitrocellulose membrane, which was subsequently blocked with 5% defatted milk for 1 hour at room temperature and coated with specific primary antibodies against β-tubulin (1:1000; Affinity, USA), BAX (1:3000; Proteintech, China), BCL-2 (1:2000; Abcam, USA), and KLF6 (1:2000; Proteintech, China) for overnight incubation at 4°C. The membrane was probed by the secondary antibody for 1 hour at 37°C. The electrochemiluminescence (ECL) kit (Affinity, USA) was dropped on the membranes and put into the chemiluminescence instrument ChemiScope 6200 Touch (Clinx, China) for automatic luminescence imaging. Image J software (version 1.53t; URL link: https://imagej.nih.gov/ij/download.html ) was used for image analysis and processing.
Bca protein assay kit
Manufactured by Coolaber
Sourced in China
The BCA Protein Assay Kit is a colorimetric detection kit used to quantify the total protein concentration in a sample. It employs the bicinchoninic acid (BCA) method, which utilizes the reduction of copper ions (Cu2+ to Cu+) by proteins in an alkaline medium, resulting in the formation of a purple-colored complex that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Bcl 2, by Abcam (1 mentions)
β tubulin, by Affinity Biosciences (1 mentions)
Bcl2 associated x (bax), by Proteintech (1 mentions)
Chemiscope 6200 touch, by Clinx (1 mentions)
Nf κb p65, by ABclonal (1 mentions)
Protein extraction kit, by Coolaber (1 mentions)
α7nachr, by ABclonal (1 mentions)
Gapdh, by ABclonal (1 mentions)
Tnf α, by ABclonal (1 mentions)
Il 10, by ABclonal (1 mentions)
Interleukin 6 (il 6), by ABclonal (1 mentions)
2 protocols using bca protein assay kit
1
Western Blot Analysis of Cell Signaling Proteins
2
Protein Expression Analysis in Lung and Gut
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The lung and gut tissues were frozen at −80°C. Proteins were extracted from lung and gut tissues using a protein extraction kit (Coolaber, China) in accordance with the manufacturer's instructions. The lung and gut tissues were homogenized on ice with RIPA buffer, and the protein concentrations were determined using a BCA protein assay kit (Coolaber, China). The protein samples were boiled in sample buffer, loaded into each lane, separated by 10% SDS-PAGE, and transferred to PVDF membranes. The membranes were washed with Tris-buffered saline with Tween 20 (TBST) three times and sealed with 5% nonfat milk for 2 h at 25°C. Subsequently, primary antibodies, including GAPDH (1:5000, ABclonal, China), MPO (1:1000, ABclonal, China), TNF-α (1:1000, ABclonal, China), IL-6 (1:1000, ABclonal, China), IL-10 (1:1000, ABclonal, China), α7nAchR (1:1000, ABclonal, China), and NF-κB p65 (1:1000, ABclonal, China) were incubated at 4°C overnight and then incubated at room temperature for 1 h. Finally, the protein levels were quantified using a chemiluminescence kit. The images were quantitatively analyzed using ImageJ analysis software.
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