SKM tissue samples were homogenized in RIPA buffer containing 50 mM Tris HCL pH 8, 150 mM NaCL, 1 % NP-40, 0.5 % sodium Deoxycholate, 0.1 % SDS and Halt protease and phosphatase inhibitor cocktail (Pierce Thermo Scientific, Rockford, IL). Samples were centrifuged at 14,000 g for 20 minutes at 4°C, and protein concentrations determined by the BCA protein assay kit (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein (30 ug) were separated by SDS page gel electrophoresis and transferred to Immobilon-PSQ transfer membrane (Millipore, Billerica, MA). TIMP-1 (LifeSpan Biosciences, Seattle, WA) and TGF-β1 (Abcam, Cambridge, MA) antibodies were used for Western blot analysis. Bands were visualized using Chemiluminescence Reagent Plus (PerkinElmer Life Science, Boston, MA), and quantified based on density with the Carestream molecular imaging software. β-actin was used as a loading control.
Chemiluminescence reagent plus
Manufactured by PerkinElmer
Sourced in United States
Chemiluminescence Reagent Plus is a laboratory reagent designed to facilitate chemiluminescence detection. It provides the necessary components to generate a chemiluminescent signal, which can be used to detect and quantify various analytes in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Semi dry transfer apparatus, by Bio-Rad (3 mentions)
Protease and phosphatase inhibitor, by Roche (2 mentions)
Protease and phosphatase inhibitor, by Merck Group (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Protein assay kit, by Bio-Rad (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Immobilon psq transfer membrane, by Merck Group (1 mentions)
Tgf β1, by Abcam (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Bio-Rad (1 mentions)
β tubulin, by Merck Group (1 mentions)
Bradford method, by Bio-Rad (1 mentions)
Immobilon, by Merck Group (1 mentions)
Goat anti rabbit igg peroxidase secondary antibody, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Hexokinase 1, by Cell Signaling Technology (1 mentions)
P8340, by Merck Group (1 mentions)
Polyvinylidene fluoride (pvdf), by Bio-Rad (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
25 protocols using chemiluminescence reagent plus
1
Quantification of TIMP-1 and TGF-β1 Proteins
2
Subcellular Protein Profiling of Mouse Neocortical Tissues
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Neocortical tissues of E14.5 mice and cultured cells were homogenized with lysis buffer containing 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 10% glycerol, and a protease inhibitor mixture (Complete; Roche). For subcellular distribution analysis, cytoplasmic and nuclear fractions of cultured NPCs were separated as described in [51] (link). The protein concentration was determined by the Bradford method (Bio-Rad). The lysates (10 µg protein per lane) were separated by 10% SDS-PAGE, electroblotted to polyvinylidene difluoride membranes (Immobilon, Merck Millipore), and incubated with primary antibodies. The primary antibodies used are: p16 (Santa Cruz F-12; 1∶200), Cdk1 (Santa Cruz sc-54; 1∶300), Sox2 (R&D Systems; 1∶300), necdin (NC243; 1∶1000) [52] (link), β-tubulin (Sigma-Aldrich; 1∶1000), Bmi1 (GBmi1; 1∶300; raised in guinea pig against GST-fused full length Bmi1), Lamin B (Santa Cruz C-20; 1∶500), Nestin (ST-1; 1∶1000), Myc (9E10; 1∶10), proliferating cell nuclear antigen (PCNA) (Santa Cruz PC10, 1∶1000), and necdin (GN1; 1∶1000). After incubation with peroxidase-conjugated IgGs (Cappel), the proteins were detected by chemiluminescence method (Chemiluminescence Reagent Plus; PerkinElmer). Signal intensities were quantified by ImageJ 1.44 software. β-tubulin was used as an internal control to normalize the expression level of each protein.
3
Extraction and detection of native membrane proteins
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Native membrane proteins were extracted from transfected cells using the ProteoExtract® Native Membrane Protein Extraction Kit (Calbiochem). Proteins from whole cells and from the membrane and soluble fractions were separated on a 12% SDS-PAGE protein gel. Part of the gel was stained with Coomassie blue and processed for mass spectrometry analysis (Pôle Protéomique, Plateforme Génomique Fonctionnelle de Bordeaux, Université de Bordeaux) and the other was transferred onto a PVDF membrane (Bio-Rad) overnight at 4 °C. To limit non-specific binding, the anti-XlEnpp4 antibody was pre-absorbed on untransfected CHO cells. The membrane was incubated overnight at 4 °C with 1:200 dilution of Enpp4 antiserum, washed and incubated in goat anti-rabbit IgG peroxidase secondary antibody (Sigma, dilution 1/2000) for 30 min at 20 °C. After several washes, immunoreactivity was detected by chemiluminescence (Western lighting Chemiluminescence Reagent Plus, Perkin Elmer).
4
Western Blot Analysis Protocol
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Cell pellets were lysed in SDS lysis buffer and sonicated, and the protein concentration of each sample was determined by using the BCA Protein Assay kit (Thermo Fisher Scientific). An equal amount of protein from each sample was loaded per lane, separated by SDS-PAGE, and then transferred onto nitrocellulose membranes. Transferred membranes were blocked with 5% non-fat milk for 1 h and then incubated with primary antibodies overnight at 4°C. On the next day, membranes were washed with TBST and incubated with the corresponding secondary antibodies for 1h at room temperature. Chemiluminescence Reagent Plus (Perkin-Elmer; Waltham, MA, USA) was used to detect signals.
5
Western Blot Protein Detection Protocol
6
Western Blot Analysis of Hexokinase Isoforms
7
Western Blot Protein Analysis Protocol
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Drug-treated cells were collected from 6 cm dishes by scraping and centrifugation. The cells were washed once with phosphate-buffered saline (PBS), and then lysed in a lysis buffer containing 1% sodium dodecyl sulfate (SDS), 10 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris-HCl (pH 8.1) and (Sigma-Aldrich, P8340). Lysates were sonicated for 15 s to shear genomic DNA and then centrifuged at 13,000 × g for 10 min. Concentrations of proteins in the supernatants were quantified using the Micro BCA Protein Assay Kit (Pierce Biotechnology, 23235). Equal amounts of each protein were resolved in a SDS-polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF, Bio-Rad, 1620184) membrane. The transblotted membrane was blocked with Tris-buffered saline containing 0.1% Tween-20 (TBST) and 5% non-fat milk for 30 min, and then incubated with the appropriate primary antibody (1:500-1: 4,000 dilution) in TBST at 4 °C overnight. The membrane was washed three times with TBST for a total of 30 min, incubated with goat anti-rabbit or anti-mouse IgG-HRP conjugates (1: 5,000 dilution) for 2 h at room temperature, and then washed again as described previously. Western Lighting Chemiluminescence Reagent Plus (Perkin-Elmer, NEL103001EA) was used to develop the images and the immunoblots were visualized by enhanced chemiluminescence.
8
Western Blot Analysis of Protein Lysates
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Total protein lysates were collected and solubilized in lysis buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors on ice for 30 min and then sonicated for 1 min. Protein concentration was determined by using BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Total proteins were separated by SDS-PAGE and then transferred onto nitrocellulose membranes. After blocking in 5% non-fat milk for 1 h, the membranes were incubated overnight at 4 °C with primary antibodies. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse antibody was applied as a secondary antibody for 1 h at room temperature. Protein detection was performed by Chemiluminescence Reagent Plus (Perkin-Elmer, Waltham, MA, USA). The relative intensity of the protein band was measured densitometrically using the Image J software. Original western blot images are found in Supplementary File S1 .
9
Protein Abundance Analysis in Tissue Lysates
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Skeletal muscle, liver, and VAD tissue lysates were prepared by dissection and homogenized in buffer (25 mM HEPES, pH 7.4; 1% Nonidet P-40; 137 mM NaCl; 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 1 μg/ml pepstatin, 5 μg/ml leupeptin) using a PRO 200 homogenizer (PROScientific, Oxford, CT, USA). The samples were centrifuged at 14,000 × g for 20 min at 4 °C, and supernatant protein concentrations were determined using a protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Supernatants (50 μg) were resolved by SDS-PAGE and subjected to immunoblotting. Protein abundance was detected with antibodies against insulin receptor-α, insulin receptor-β, Akt1, phospho-Akt1, PDK1, phospho-PDK1, GSK3β, phospho-GSK3β, and β-actin. All proteins were detected with HRP-conjugated secondary antibodies using Chemiluminescence Reagent Plus (PerkinElmer Life Science, Boston, MA, USA), and quantified with a densitometer. All proteins were normalized by β-actin, and specific protein phosphorylation was normalized by corresponding proteins.
10
Protein Expression Analysis of DNA Repair Enzymes
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Cells were harvested and lysed with standard PLC lysis buffer containing protease and phosphatase inhibitors (Sigma‐Aldrich, USA). Then the lysate was centrifuged to collect supernatant. Protein concentration was determined using BCA assay (Thermo‐Fisher Scientific, USA). Finally, equivalent amounts of protein (30 μg) were separated with 7.5%–12.5% SDS‐PAGE gel with electrophoresis, then were electro‐transferred onto PVDF membrane (NEN Research Products, USA). Membrane was soaked in 5% nonfat milk for 2 h at room temperature. The primary antibodies were applied at dilutions of 1:1000 for anti‐ALKBH7 (A2331, ABclonal, China), anti‐Alkylpurine‐DNA‐N‐glycosylase (APNG) (ab155092, Abcam, UK), MGMT (Proteintech, 17195‐1‐AP, China), or anti‐γ‐H2AX (97148SF, CST, USA), respectively, at 4°C overnight. After incubation with the primary antibody, the membrane was washed three times with PBS with 0.1% Tween 20 for 10 min, then incubated with the second antibody specific for the primary antibody (BioRad Laboratories, USA). The binding was visualized with Chemiluminescence Reagent Plus (PerkinElmer, USA) and analyzed in ImageJ.
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