Analysis of oxidized proteins was performed by western blot in liver homogenates using an Oxyblot kit (Millipore Bioscience Research Reagents, Temecula, CA). The same amounts of liver proteins (35 μg) were reacted with dinitrophenylhydrazine (DNP) for 20 min, followed by neutralization with a solution containing glycerol and 2-mercaptoethanol, resolved in 12.5% sodium dodecyl sulphate–polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, blocked with nonfat milk, and incubated with a rabbit anti-DNP antibody (1: 150) at 4 °C overnight. After washing, the membrane was incubated with the secondary antibody (1:300) conjugated to horseradish peroxidase and detected by a chemiluminescence detection kit (Cell Signaling Technology Inc., Danvers, MA). Reactive bands were visualized by the enhanced chemiluminescence method on a VersaDoc Image System (Bio-Rad Laboratories, Hercules, CA).
Versadoc image system
Manufactured by Bio-Rad
Sourced in United States
The VersaDoc Image System is a versatile imaging device designed for capturing high-quality digital images of various biological samples, such as gels, blots, and plates. The system utilizes a charged-coupled device (CCD) camera to provide accurate and sensitive image acquisition, enabling researchers to document and analyze their experimental results effectively.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Chemiluminescence detection kit, by Cell Signaling Technology (2 mentions)
Immuno star hrp chemiluminescent kit, by Bio-Rad (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Dynabeads protein g immunoprecipitation kit, by Thermo Fisher Scientific (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Anti wee1, by Cell Signaling Technology (1 mentions)
Anti ccnb1, by Santa Cruz Biotechnology (1 mentions)
Western lightning chemiluminescence reagent plus kit, by PerkinElmer (1 mentions)
Immobilon p transfer membrane, by Merck Group (1 mentions)
Hpa014711, by Merck Group (1 mentions)
Immun star hrp chemiluminescent kit, by Bio-Rad (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Criterion xt, by Bio-Rad (1 mentions)
Xt sample buffer, by Bio-Rad (1 mentions)
Bio safe coomassie g 250, by Bio-Rad (1 mentions)
Ecl kit, by Solarbio (1 mentions)
Ab31013, by Abcam (1 mentions)
Ab2185, by Abcam (1 mentions)
Zb 2301, by ZSGB-BIO (1 mentions)
11 protocols using versadoc image system
1
Quantification of Oxidized Proteins in Liver
2
Western Blot Analysis of GRP78 Protein
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Cell extracts were prepared as described previously19 (link) and the protein content was determined by the Bradford assay. Briefly, a total of 30 µg of total protein was added to each well of 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Electrophoresis was carried out. Then, proteins on the gels were transferred onto nitrocellulose membranes. Membranes were blocked using 5% skim milk before overnight incubation with the primary antibodies at a concentration of 1:100 v/v. Incubation of membranes with horseradish peroxidase–conjugated goat antirabbit IgG or goat antimouse IgG (1:2,000 v/v; Bio-Rad Laboratories Inc., Hercules, CA, USA) was performed at room temperature with shaking for 1 hour. Labeled bands were detected with Immuno-Star HRP Chemiluminescent kit and the images were captured and quantified using the VersaDoc image system (Bio-Rad). The relative expression of detected proteins was determined as shown previously.20 (link) Briefly, the relative expression of GRP78 was determined by dividing the densitometric value of GRP78 by that of the glyceraldehyde-3-phosphate dehydrogenase.
3
Cell Lysate Preparation and Western Blotting
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To prepanre cell lysates, RIPA Lysis and Extraction Buffer (Invitrogen; ThermoFisher Scientific, Inc., MA, USA) which contained 10% protease inhibitor (Thermo Scientific, USA) were used to extract cell lysates by incubated on ice for 30 min, and then centrifuged at 14000 x g for 15 min at 4 °C. The protein concentrations were determined by BCA kit (Thermo Scientific, USA). For western blotting, we used the protocol which was performed previously [31 (link)]. In brief, we first mixed supernatants with 4x SDS-PAGE sample loading buffer, and they were denatured at 95 °C for 10 min. Second, the proteins were separated by SDS-PAGE gel, transferred to a polyvinylidene difluoride membranes, incubated with specific primary antibodies at 4 °C overnights, and detected with horseradish peroxidase (HRP)-conjugated secondary antibodies by using a VersaDoc Image System (BioRad, Hercules, CA, USA). For immunoprecipitation, the lysate was treated using the Dynabeads™ Protein G Immunoprecipitation Kit (Invitrogen; ThermoFisher Scientific, Inc., MA, USA) according to the protocol. The final precipitated proteins were analyzed via western blotting with the corresponding antibodies.
4
Gel Image Analysis Protocol
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The gel images were analysed with a Versa doc image system (Bio-Rad) and PD Quest software version 8.0.1 (Bio-Rad). Spot detection was performed by matching the gels automatically with manual verification. The spots detected in at least two replicate gels were selected for annotation.
5
Oxidized Proteins Analysis in Liver Mitochondria
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The analysis of oxidized proteins was performed by western blot in liver mitochondria using an Oxyblot kit (Millipore Bioscience Research Reagents, Temecula, CA, USA) [16 (link)]. The same amounts of mitochondrial proteins (35 µg) were reacted with dinitrophenyl hydrazine (DNPH) for 20 min, followed by neutralization with a solution containing glycerol and 2-mercaptoethanol, resolved in 12% SDS-polyacrylamide gel electrophoresis. After the transfer to a nitrocellulose membrane, a blocking step with non-fat milk and incubation with a rabbit anti-DNPH antibody (1: 150) at 4 °C overnight followed. After washing, the membrane was incubated with the secondary antibody (1:300) conjugated to horseradish peroxidase and detected by a chemiluminescence detection kit (Cell Signaling Technology Inc., Danvers, MA, USA). Reactive bands were visualized by the enhanced chemiluminescence method on a VersaDoc Image System (Bio-Rad Laboratories, Hercules, CA, USA). Band density was determined with TotalLab software. The test provides a qualitative analysis of the total proteins’ oxidation state change.
6
Quantitative Western Blot Analysis
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Cell lysates were prepared with RadioImmuno Precipitation Assay buffer (Upstate). Lysates containing equal amounts of protein were separated by SDS-PAGE and electroblotted onto Immobilon™-P Transfer Membrane (Millipore). The filters were individually probed with specific primary antibody. Protein bands were detected by the Western Lightning Chemiluminescence Reagent Plus Kit (Perkin-Elmer Life Sciences) with horseradish peroxide labeled secondary antibody as suggested by the manufacturer and visualized on a VersaDoc Image System (Bio-Rad). The intensity of bands was quantified by densitometry and normalized to ACTB in each lane. Anti-human EMP2 (1:100, HPA014711, Sigma-Aldrich), anti-cyclin dependent kinase 1 (CDK1; 1:200, sc-54, Santa Cruz), anti-pCDK1(Y15) (1:250, BD Transduction Laboratories™), anti-WEE1 (1:1000, #4936, Cell Signaling), anti-CCNB1 (1:500, sc-245, Santa Cruz), anti-pCDC25C(S216) (1:1000, #4901, Cell Signaling) and anti-pCREB1(S133) (1:200, sc-7978, Santa Cruz) were used as primary antibodies for immunoblotting analysis and anti-ACTB (1:3000, Chemicon) was served as a loading control.
7
Cell Lysis and Protein Aggregation Analysis
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The cell lysates were extracted by lysis buffer (containing 50 mM HEPES, pH 7.6, 150 mM NaCl, 1% Triton X-100, 10 mM NaF, 20 mM Na4P2O7, 20 mM β-glycerol phosphate, 1 mM Na3VO4, 10 mg/ml leupeptin, 10 mg/ml aprotinin, and 1 mM PMSF), incubated on ice for 20 min, and then centrifuged at 14000 × g for 10 min at 4°C. The supernatants were mixed with equal volume of 2x SDS-PAGE sample loading buffer and then denatured at 100°C for 10 min. The proteins were separated by SDS-PAGE gel, transferred to a nitrocellulose membrane, incubated with specific primary antibodies for overnight, and detected with horseradish peroxidase (HRP)-conjugated secondary antibodies by using a VersaDoc Image System (BioRad, Hercules, CA, USA).
Protein aggregation was determined using ProteoStatTM Protein Aggregation Assay Kit (ENZ-51023-KP002) from Enzo Life Sciences International Inc. (Plymouth Meeting, PA, USA), following the manufacturer’s instruction.
Protein aggregation was determined using ProteoStatTM Protein Aggregation Assay Kit (ENZ-51023-KP002) from Enzo Life Sciences International Inc. (Plymouth Meeting, PA, USA), following the manufacturer’s instruction.
8
Protein Quantification and Western Blotting
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The protein content of cell extracts was determined by a Bradford assay (Bio-Rad, Hercules, CA, USA). A total of 20–30 μg protein was electrophoresed by 10–15% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked and incubated with the primary antibodies at the appropriate concentrations, and subsequently incubated with HRP-conjugated goat anti-rabbit or goat anti-mouse IgGs (1:2,000; Beyotime Institute of Biotechnology). Labeled bands were detected by the Immun-Star™ HRP Chemiluminescent kit (Bio-Rad), and images were captured and the intensity of the bands was quantified by the VersaDoc™ image system (Bio-Rad, Regents Park, Australia).
9
Protein Profiling of Milk Fat Globule Membrane
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The protein profile was investigated by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Samples were normalized according to protein content (4 mg/mL) and then dissolved in sample buffer (4× XT Sample Buffer, Bio-Rad, Hercules, CA, USA) containing 5mL/100mL of Tris (2-carboxyethyl) phosphine hydrochloride (TCEP.HCl). The mix, with a final protein concentration of 1mg/mL, was heated at 95 °C for 3 min and then loaded on a 12% Bis-Trispolyacrilamide gel (Criterion_XT, Bio-Rad). Moreover, the protein pattern of MFGM isolated from selected BM and BS was investigated by means of 3–8% Tris-Acetate gels (Criterion_XT, Bio-Rad), which allowed for better separation and discrimination of high-molecular-weight proteins (50–250 kDa). Electrophoresis was conducted for 1 h at a constant voltage of 150 V. Finally, the 2-D gels were stained with a dye (Bio-Safe™ Coomassie G-250, Bio-Rad) and scanned with a Versa-Doc image system (Bio-Rad).
10
Protein Expression Analysis Protocol
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Proteins were extracted and electroblotted as previously described.34 The primary antibodies anti‐HIF‐1α (ab2185, 1:1000), anti‐SLIT3 (R&D System, MN, USA, 1:200), anti‐ALP (ab17973, 1:1000), anti‐RUNX2 (ab23981, 1:1000) and anti‐OCN (ab93876, 1:1000) were added to the strip at 4°C overnight after blocked with 5% BSA (ZSGB‐BIO), the secondary horseradish peroxidase antibody (Solarbio) was then applied to the section for an hour. Finally, labelled membranes were identified by ECL kit (Solarbio) and were assessed by Bio‐Rad VersaDoc image system (Bio‐Rad Laboratories); all primary antibodies used in current study were from Abcam unless otherwise specified.
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