Cell pellets were homogenized and lysed in T-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA) with the HaltTM Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific) at a ratio of 100:1. The Pierce BCA Protein Assay Kit (ThermoFisher Scientific) was used to determine the protein concentration. Protein denaturation was done in sample buffer (Laemmli Sample buffer: β-ME = 19:1) at 95 °C for 5 min. Proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gel was placed onto a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was blocked in EveryBlot Blocking Buffer (Bio-Rad Laboratories) at room temperature for 15 min and subsequently incubated with primary antibodies (Table 1 ) at 4 °C overnight. The next day, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Table 1 ) at room temperature for 1 h. After washing with Tris Buffered Saline with Tween® 20 (Cell Signaling Technology, Inc., Cambridge, UK), membranes were developed with the Clarity Western ECL Substrate (Bio-Rad Laboratories). Image acquisition was performed on a ChemiDocTM MP Imaging System (Bio-Rad Laboratories).
T per mammalian protein extraction reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
T-PER Mammalian Protein Extraction Reagent is a complete, ready-to-use solution for the extraction of cellular proteins from mammalian cell and tissue samples. It is designed to effectively solubilize and extract proteins for further analysis.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Tris buffered saline with tween 20, by Cell Signaling Technology (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Everyblot blocking buffer, by Bio-Rad (1 mentions)
Bicinchoninic acid protein assay, by Thermo Fisher Scientific (1 mentions)
Cocktail of protease and phosphatase inhibitors, by Thermo Fisher Scientific (1 mentions)
C digit blot scanner, by LI COR (1 mentions)
Image studio software, by LI COR (1 mentions)
Phosphatase inhibitors, by Cayman Chemical (1 mentions)
Anti β actin, by Thermo Fisher Scientific (1 mentions)
Anti rabbit igg horseradish peroxidase, by Thermo Fisher Scientific (1 mentions)
Clarity western ecl luminol substrate, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail and phosphatase inhibitor cocktail 2 and 3, by Merck Group (1 mentions)
5 protocols using t per mammalian protein extraction reagent
1
Western Blot Protein Analysis Protocol
2
Adipose Tissue Protein Extraction and Analysis
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Proteins were isolated from visceral adipose tissues using T-PER Mammalian Protein Extraction Reagent (Thermo Scientific) as indicated by the manufacturer, in the presence of a cocktail of protease and phosphatase inhibitors (Thermo Scientific). Protein content was determined by the bicinchoninic acid protein assay (Thermo Scientific) and 70 μg of proteins were separated with SDS-PAGE under reducing conditions. The separated proteins were then electrophoretically transferred to a nitrocellulose membrane (BioRad). Proteins of interest were revealed with specific antibodies: Ob Antibody (H-146) sc-9014 (Santa Cruz) and anti-β actin (Sigma-Aldrich). The immunostaining was detected using horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin (Novex), bands were revealed by the SuperSignal Substrate (Pierce), detected using the C-Digit Blot Scanner and quantified by densitometry using the Image Studio software (all from Li-Cor).
3
BDNF Protein Quantification in Hippocampus and PFC
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The hippocampus and prefrontal cortex samples were homogenized in T-PER mammalian protein extraction reagent (Thermo Fisher Scientific, Waltham, MA, USA) with protease (Merck Millipore, Burlington, MA, USA) and phosphatase inhibitors (Cayman Chemical, Ann Arbor, MI, USA). The concentration of proteins was determined using the Bradford assay. The aliquots (40 μg) were solubilized in Laemmli buffer with 2% 2-mercaptoethanol and were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anti-BDNF (15kDa, 1:500 dilution) and anti-β-actin (1:1000 dilution) (Thermo Fisher Scientific) were used as primary antibodies, while anti rabbit IgG (horse radish peroxidase) (1:2000 dilution; Thermo Fisher Scientific) was used as the secondary antibody. Proteins were detected using Clarity Western ECL Luminol Substrate (Bio-Rad, Hercules, CA, USA). Chemi Doc Camera with Image Lab 5.2.1 software (Bio-Rad) was used to quantify the integrated optical density of the bands.
4
Muscle Protein Expression Analysis
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The peripheral parts of muscle specimens were immediately frozen in liquid nitrogen and stored at −80°C for biochemical studies. Western blotting was performed as previously described (Fujimura and Usuki, 2015 (link), 2017 (link)). Briefly, the samples were sonicated for 5 s in tissue lysis buffer (T-PER Mammalian Protein Extraction Reagent; Pierce Biotechnology, Rockford, USA) containing Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail 2 and 3 (Sigma-Aldrich, St Louis, USA). The samples were centrifuged (14,000 g for 1 h), and the supernatants were collected. The protein content was determined using the DC Protein Assay Kit II (Bio-Rad Laboratories, Hercules, USA). The cell lysates (20 μg protein) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% gel (Tefco, Tokyo, Japan) and transferred to nitrocellulose membranes (GE Healthcare, Buckinghamshire, UK). The membranes were then subjected to the following antibody probes: anti-MGF (Millipore, Billerica, USA); anti-IGF-I (Santa Cruz Biotechnology, CA, USA); anti-YAP1 (Novus Biologicals, Centennial, CO, USA); anti-phospho-YAP1 (Abcam, Cambridge, UK); anti-PAX7 (Cytoskeleton Incorporated, Denver, USA); anti-phospho-ERK, anti-ERK, anti-AKT, anti-phospho-AKT, anti-phospho-4EBP1, and anti-phospho-p70 S6 kinase (Cell Signaling Technologies); and anti-β-actin (Sigma-Aldrich).
5
Protein Quantification and Cytokine Analysis
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Proteins were extracted using T-PER Mammalian Protein Extraction Reagent (Pierce Biotechnology, Rockford, IL). C1q/TNF-related protein 9 (CTRP9) and TNF-α were measured using the Mouse CTRP9 ELISA kit (Aviscera Bioscience, Santa Clara, CA) and the Mouse TNFα ELISA kit (Thermo Scientific, Seattle, WA), respectively. The total protein concentrations were measured by the Pierce BCA Protein Assay kit (Thermo Scientific). Individual protein concentrations were calculated as the abundance of specific protein constituents divided by the total protein concentrations. Serum IL-6 level was determined by using a commercial ELISA kit (Thermo Scientific).
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