Proteins were collected from human mesenteric preadipocytes of control, UC and CD patients (n=4 per group) in RIPA TRITON X100 (Boston Bioproducts BP-116TX) with protease and phosphatase inhibitors (Sigma-Aldrich). 30µg of protein were loaded on a 10% polyacrylamide gel and electrophoresed for 1.5 hrs. The proteins were transferred on PVDF membranes, and membranes were blocked for 1 hr at RT in LI-COR blocking buffer (LI-COR Biosciences, Lincoln, NE). The membranes were blotted with a rabbit NK-1R primary antibody, at a dilution of 1:100, O/N at RT (Santa Cruz Biotechnology Inc, sc-15323, Santa Cruz, CA). Secondary goat anti-rabbit antibody (1:15000, LI-COR Biosciences, cat# 926-32211) was added for 1 hr at RT. Loading was normalized using a mouse β-actin primary antibody (1:1000, Santa Cruz Biotechnology Inc., cat# sc-81178) and a goat anti-mouse secondary antibody (1:15000, LI-COR Biosciences #926-68170). Bands were visualized and quantified using Odyssey IR Imaging System (LI-COR Biosciences).
Ripa triton x100
Manufactured by Boston BioProducts
RIPA TRITON X100 is a lysis buffer used for the extraction and solubilization of proteins from cells and tissues. It is a non-ionic detergent solution that aids in the disruption of cell membranes and the release of intracellular proteins.
Automatically generated - may contain errors
Lab products found in correlation
Protease and phosphatase inhibitor, by Merck Group (2 mentions)
Blocking buffer, by LI COR (2 mentions)
Odyssey ir imaging system, by LI COR (2 mentions)
Goat anti mouse secondary antibody, by LI COR (1 mentions)
Sc 15323, by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse secondary antibody, by Santa Cruz Biotechnology (1 mentions)
2 protocols using ripa triton x100
1
Western Blot Analysis of NK-1R in Preadipocytes
2
Quantifying NK-1R Expression in Mesenteric Preadipocytes
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Proteins were collected from human mesenteric preadipocytes of control, UC, and Crohn’s disease patients (n = 4 per group) in RIPA TRITON X100 (BP-116TX; Boston BioProducts, Ashland, MA) with protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO). We loaded 30 μg of protein on a 10% polyacrylamide gel and electrophoresed it for 1.5 hours. The proteins were transferred on polyvinylidene fluoride membranes, and the membranes were blocked for 1 hour at room temperature in LI-COR blocking buffer (LI-COR Biosciences, Lincoln, NE). The membranes were blotted with a rabbit NK-1R primary antibody at a dilution of 1:100 overnight at room temperature (sc-15323; Santa Cruz Biotechnology, Santa Cruz, CA). Secondary goat anti-rabbit antibody (1:15,000, cat. no. 926-32211; LI-COR Biosciences) was added for 1 hour at room temperature. Loading was normalized using a mouse β-actin primary antibody (1:1000, cat. no. sc-81178; Santa Cruz Biotechnology) and a goat anti-mouse secondary antibody (1:15,000, cat. no. 926-68170; LI-COR Biosciences). Bands were visualized and quantified using the Odyssey IR Imaging System (LI-COR Biosciences).
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