Western blotting was performed as previously described [16] (link), [20] (link). Briefly, the EDL muscle was homogenized and the total protein lysates analyzed for expression of markers for protein degradation using antibody against Ubiquitin and p70 (total and phosphorylated), respectively (1:500, Santa Cruz Biotechnology, Santa Cruz, CA). The blots were incubated overnight at 4 oC followed by incubating with peroxidase conjugated secondary antibody (1:5000 dilution) and immunodetection assessed with the Enhanced Chemiluminescence Prime Western Blot Detection Reagent (Amersham, Piscataway, NJ). Band intensity was analyzed by ChemiDoc MP Imaging System (Imaging Lab 4.0, Bio-Rad, Richmond, CA) and normalized to total protein expression using Ponceau S (Santa Cruz Biotechnology, Santa Cruz, CA) or phosphorylated to total p70 were utilized as the normalization for p70 expression.
Enhanced chemiluminescence prime western blot detection reagent
Manufactured by Cytiva
Sourced in United States
The Enhanced Chemiluminescence Prime Western Blot Detection Reagent is a laboratory product used for the detection and visualization of proteins in Western blot analysis. It generates a chemiluminescent signal that can be captured and quantified to determine the presence and abundance of specific proteins in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc mp imaging system, by Bio-Rad (5 mentions)
Ponceau s, by Santa Cruz Biotechnology (3 mentions)
Cayman s nuclear extraction kit, by Cayman Chemical (2 mentions)
Bullet blender, by Next Advance (1 mentions)
Antibody against nox4, by Novus Biologicals (1 mentions)
Antibody against hif 1α, by Novus Biologicals (1 mentions)
5 protocols using enhanced chemiluminescence prime western blot detection reagent
1
Protein Degradation Markers Analysis via Western Blotting
2
Analyzing β-Catenin Signaling in Bone
3
Quantifying Mitochondrial Proteins by Western Blot
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Western blotting was performed as previously described39 (link) using the EDL and soleus lysates stored at − 20 °C. Protein expression was assessed via commercially available monoclonal antibodies for the following: electron transport chain protein (rodent OXPHOS cocktail, Abcam, ab110413, Cambridge, UK, 1:500 dilution) and VDAC 2 (voltage-dependent anion channel, Cell Signaling, #9412, Danvers, MA, 1:500 dilution). The primary antibody was maintained at 4 °C overnight followed by incubating with peroxidase conjugated secondary antibody (1:5000 dilution), and immunodetection with the Enhanced Chemiluminescence Prime Western Blot Detection Reagent (Amersham, Piscataway, NJ). The band intensity was analyzed by ChemiDoc MP Imaging System (Imaging Lab 4.0, Bio-Rad, Richmond, CA) and normalized to total protein expression using Ponceau S (Santa Cruz Biotechnology, Santa Cruz, CA).
4
Measuring Oxidative Stress Markers
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Western blotting was performed as previously described [19 (link)]. In brief, the EDL from NL and CKD were homogenized and the total tissue protein lysates were stored at -20C. The expression of Nox4 was measured using antibody against Nox4 (1:300 dilution; Novus Biologicals, Littleton, CO). Nuclear and cytosolic protein was isolated using Cayman’s Nuclear Extraction kit (Cayman Chemical Company, Ann Arbor, MI) according to the manufacturer’s instructions. Nrf2 was measured in the nuclear fraction, and the major regulator of Nrf2, Keap1 (Kelch-like ECH-associated protein I) was measured in the cytosolic fraction with antibody against Nrf2 or Keap 1(1:1000, Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C followed by incubating with peroxidase conjugated secondary antibody (1:5000 dilution), and immunodetection with the Enhanced Chemiluminescence Prime Western Blot Detection Reagent (Amersham, Piscataway, NJ). The band intensity was analyzed by ChemiDoc MP Imaging System (Imaging Lab 4.0, Bio-Rad, Richmond, CA) and normalized to total protein expression using Ponceau S (Santa Cruz Biotechnology, Santa Cruz, CA).
5
Isolation and Analysis of MSC Nuclear and Cytosolic Proteins
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Nuclear and cytosolic protein from cultured MSC was isolated using Cayman's Nuclear Extraction kit (Cayman Chemical Company, Ann Arbor, Mich., USA) according to the manufacturer's instructions. The expression of HIF-1α and Nrf2 were measured in the nuclear fraction, and the major regulator of Nrf2, Keap1 (Kelchlike ECH-associated protein I) was measured in the cytosolic fraction by Western blot. Briefly, 30 μg of nuclear or cytosolic protein was loaded on 10% SDS-PAGE and the blots were incubated with antibody against HIF-1α (1: 1,000, Novus Biologicals, Littleton, Colo., USA), Nrf2 or Keap1 (1: 1,000, Santa Cruz Biotechnology, Santa Cruz, Calif., USA) overnight at 4 ° C followed by incubating with peroxidase conjugated secondary antibody (1: 5,000 dilution), and immunodetection with the Enhanced Chemiluminescence Prime Western Blot Detection Reagent (Amersham, Piscataway, N.J., USA). The blots were also stained for Ponceau S staining as loading control [31] . The band intensity was analyzed by ChemiDoc MP Imaging System (Imaging Lab 4.0, Bio-Rad, Richmond, Calif., USA) and normalized by Ponceau S staining.
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