Ipsilateral and contralateral hippocampi were homogenized in 250 μl RIPA buffer (Pierce) in the presence of proteinase inhibitor cocktail P8340 (Sigma-Aldrich, St. Louis, MO). Lysates were centrifuged at 16,000 g for 10 min at 4°C. The supernatant was collected, and the protein concentration was measured using Bradford assay kit (Bio-Rad, Hercules, CA). Fifty μg of total proteins of each sample was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The resolved proteins were transferred to nitrocellulose membranes. The membranes were incubated overnight at 4°C with anti-KMO (#10698, Proteintech, Rosemont, IL) diluted 1/1,000 in a 5 % of blocking buffer solution, followed by horseradish peroxidase–conjugated goat anti-rabbit secondary antibody (#111-035-144, Jackson ImmunoResearch, West Grove, PA) diluted 1/4,000 for 1 h at room temperature. The protein band was revealed with an ECL (GE Healthcare), and the protein band intensity was quantified by using the ImageQuant LAS4000 (GE Healthcare). The results were normalized for β-actin (#A3854, Sigma Aldrich, St Louis, MO diluted 1/5,000 in blocking buffer) used as a protein loading control.
Proteinase inhibitor cocktail p8340
Manufactured by Merck Group
Proteinase inhibitor cocktail P8340 is a mixture of various proteinase inhibitors designed to inhibit a broad spectrum of proteolytic enzyme activities. It is commonly used in biochemical and cell biology applications to prevent protein degradation during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant las 4000, by GE Healthcare (1 mentions)
Bradford assay kit, by Bio-Rad (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
β actin, by Merck Group (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions)
2 protocols using proteinase inhibitor cocktail p8340
1
Western Blot Analysis of KMO Levels
2
Intestinal Mucosa Protein Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Intestinal sections of around 2 cm were thawed onto an ice-cold plate and opened by means of a longitudinal cut. All process was carried out at 4°C. Luminal surface was thorough cleaned using sterile gauze and PBS to eliminate mucus and blotted dry onto dried gauze. Mucosa scrapings were obtained using a razor, weighted and homogenized in lysis buffer (10 mM Tris-HCl, pH 7.6; 1 mM EDTA, 0.25 M sucrose; 1:100 (vol/vol) Proteinase Inhibitor Cocktail P8340 (Sigma-Aldrich) using pre-chilled mortar and pestles. Lysis buffer was used in a proportion of 1 ml per 250 mg of mucosa. Homogenized samples were incubated for 20 min in an orbital shaker and finally centrifuged at 10,000 × g for 30 min at 4°C. The supernatant was stored at −80°C until use. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Cultek SL, Madrid, Spain) using BSA as standard according to manufacturer's instructions.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!