HepG2 were seeded at a density of 1 × 106 in 60 mm dish and allowed to attach overnight. Cells were treated without or with 30 μg of extract for 6, 12, 24, 48 h. Cells were lysed and total protein was quantified using BCA. 20 μg of total protein was separated on SDS-PAGE, transferred onto nitrocellulose membranes that were blocked using 5% BSA TBST. Primary antibodies against Cdc2 (1:000; cell signalling), p-Cdc2 (1:1000; cell signalling), Cyclin B1 (1:1000; cell signalling), Cyclin A1 (1:000; Abcam) were used. GAPDH (1:15000; Abcam) was used as loading control. Proteins were detected using LI-COR C-DiGit Chemiluminescence Western Blot Scanner.
C digit chemiluminescence western blot scanner
Manufactured by LI COR
Sourced in United States
The C-DiGit Chemiluminescence Western Blot Scanner is a laboratory equipment used for the detection and quantification of chemiluminescent signals in Western blot analysis. It captures digital images of chemiluminescent blots and provides accurate and reproducible data.
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Lab products found in correlation
Protean i12 ief system, by Bio-Rad (2 mentions)
Dctm protein assay, by Bio-Rad (2 mentions)
Immun star hrp substrate, by Bio-Rad (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Cyclin a1, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Enhanced chemiluminescence reagent, by GE Healthcare (1 mentions)
Blocking reagent, by GE Healthcare (1 mentions)
Ab11433, by Abcam (1 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions)
Protease inhibitor cocktail, by AppliChem (1 mentions)
Ripa lysis buffer, by Bio-Rad (1 mentions)
Mouse monoclonal anti flag m2 antibody, by Merck Group (1 mentions)
Luminata forte western hrp substrate, by Merck Group (1 mentions)
Western blotting luminol reagent, by Santa Cruz Biotechnology (1 mentions)
Pvdf blotting membrane, by GE Healthcare (1 mentions)
Gtx121929, by GeneTex (1 mentions)
Pa5 20812, by Thermo Fisher Scientific (1 mentions)
Blotting grade blocker, by Bio-Rad (1 mentions)
16 protocols using c digit chemiluminescence western blot scanner
1
Regulation of Cell Cycle Proteins
2
Western Blot Analysis of Key Regulatory Proteins
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The cells samples lysates were extracted with lysis buffer composed of 50 mM Tris, pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 10 mM sodium phosphate, and a complete tablet protease Inhibitor Cocktail per 100 ml of buffer, and the protein concentration in the lysates was quantified using an enhanced bicinchoninic acid protein assay kit with bovine serum albumin as a standard. The total protein extract will be used for western blot analysis. Equal amounts of total protein were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred into a nitrocellulose membrane, followed by incubation overnight to 4°C using the following dilution of primary antibodies: anti-p53 (1:100), anti NQO1 (1:1000), anti-MDM2 (1:500), anti-E6AP (1:1000), anti-lamin A/C (1:500), anti-GAPDH (1:1000) and following by incubation with secondary antibody in blocking solution 1 h room temperature; anti-mouse (1:10000), anti-goat (1:20000) finally protein expression levels were visualized with Li-COR C-DiGit chemiluminescence western blot scanner and UVP Imaging system.
3
Western Blot Analysis of Muscle Proteins
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The dissected soleus muscle was weighted, homogenised in lysis buffer containing 10 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, and protease inhibitors. After centrifugation, protein samples were heat denatured at 96°C for 5 minutes. Samples (10 μg/lane) were separated by SDS-PAGE and were then transferred to a nitrocellulose membrane (GE Healthcare) for 1 hour at room temperature and blocked overnight at 4°C in 2% blocking reagent (GE Healthcare) in Tris-buffered saline buffer with 0.1% Tween 20. Immunoblotting was performed using a mouse monoclonal anti-mTOR (#2972); AKT (#9272), p-mTOR (#2971), LC3 (#12741), p62 (#5114), and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) thermo scientific AM4300 were purchased from Cell Signalling Co., Ltd.) with dilution 1:1000. The signals were developed using enhanced chemiluminescence reagent (GE Healthcare) and imaged (LI-COR C-DiGit Chemiluminescence Western Blot Scanner). The band intensities were determined using ImageJ Software (NIH). Blots were stripped using stripping buffer from thermo scientific according to manufacturer protocols and reprobed using with an antiGAPDH as internal control to monitor the level of protein.
4
Western Blot Analysis of Autophagy Proteins
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For Western blot analysis, the hypothalamus and striatum were quickly dissected from mouse brain. Whole striatal and hypothalamic lysates were prepared on ice in RIPA lysis buffer (ATTO, Tokyo, Japan) supplemented with protease inhibitor cocktail (ATTO) and phosphate inhibitor cocktail (ATTO). Whole lysates were homogenized in ice-cold buffer and sonicated for 10 sec. Protein concentrations were estimated using a DCTM protein assay (Bio-Rad, Hercules, CA) and 20 μg of each proteins were subjected to a 15% sodium dodceyl sulfate-polyacrylamide gel electrophoresis and transferred with EzFastBlot transfer buffer (ATTO) to polyvinylidene difluoride (PVDF) membrane (ATTO). Protein bands were visualized using C-DiGit Chemiluminescence Western Blot Scanner (Li-COR Biosciences). Band intensity was quantitated and normalized using α-tubulin as an internal control. In addition, change in the expression level of autophagy related proteins was further analyzed with two-dimensional gel electrophoresis using a PROTEAN i12 IEF System (Bio-Rad).
5
Analysis of Autophagy Proteins in RF-Exposed Mice
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Sham-or RF-exposed mice were quickly sacrificed and the cerebral cortex was rapidly dissected from each brain. Some tissue was lysed with RIPA lysis buffer (ATTO, Japan) supplemented with protease inhibitor and phosphate inhibitor cocktail (ATTO). Lysates were homogenized and sonicated briefly on ice. Concentration of proteins was estimated using a DCTM protein assay (Bio-Rad, USA). Total protein (50–100 μg) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the resolved proteins were transferred with EzFastBlot transfer buffer (ATTO) to a polyvinylidene difluoride (PVDF) transfer membrane (ATTO). Protein bands were visualized using C-DiGit Chemiluminescence Western Blot Scanner (Li-Cor). The intensity of band was quantified and normalized using α-tubulin as an internal control. Alterations in the expression level of autophagy-related proteins was further analysed with two-dimensional (2D) gel electrophoresis using a PROTEAN i12 IEF System (Bio-Rad).
6
VCP/p97 Antibody Western Blotting
7
MITF Protein Expression Verification
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The MITF protein in transfected G361 cells was verified by western blot analysis using a mouse monoclonal anti-Flag M2 antibody. Cells cultured for 48 h were harvested and protein isolated using RIPA lysis buffer (Bio-Rad), supplemented with protease inhibitor cocktail (AppliChem, Germany) and 1 mM phenylmethylsulfonyl fluoride (Merck, Germany). The proteins (20 µg) were then subjected to SDS-PAGE (12%) and transferred onto a polyvinylidene fluoride membrane with pore size 0.22 µm. The membrane was blocked using blocking solution (3% bovine serum albumin in TBS plus 0.1% Tween-20) for 2 h and then stained with a mouse monoclonal anti-Flag M2 antibody (1:1,000 dilution, Sigma) at 4 °C overnight. A goat anti-mouse GAPDH antibody (Thermo Scientific) was used as a control for protein loading. After washing with washing buffer (TBS plus 0.1% Tween-20) for 15 min, four times, the membrane was stained for 1 h with a goat anti-mouse IgG secondary antibody conjugated with horseradish peroxidase (1:1,000 dilution, Thermo Fisher Scientific) at room temperature followed by four washes. Protein levels were measured using Luminata Forte Western HRP substrate (Merck, USA) with a C-DiGit Chemiluminescence Western Blot Scanner (LI-COR Bioscience).
8
Quantifying Angiotensin II and NADPH Oxidase Proteins
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For determining the expression of angiotensin II receptor (AT1-R) and NADPH oxidase 4 (NOX-4) proteins a Western blot technique was employed. For that purpose, 100 mg of kidney tissue were homogenized with lysis buffer supplemented with complete™ Mini Protease Inhibitor Cocktail, Roche Diagnostics, Germany). Then, homogenates were sonicated during 2 minutes, at 4 °C, and were centrifuged at 10,956 g during 10 minutes, at 4 °C. Protein concentration in the supernatant was determined with Bradford Protein Assay Kit (Bio- RAD, USA). 50 μg of protein were separated by dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE, transferred to a PVDF Blotting Membrane (GE Healthcare Life Science, Germany) in a semi-dry system. Membranes were blocked with Blotting-Grade Blocker (Bio-RAD, USA), for 60 min at room temperature. Incubation was done with primary antibodies against AT1-R and NOX-4 (PA5-20812 Thermo Fisher, USA and GTX121929, GeneTex Inc, USA) overnight at 4 °C. Then a secondary antibody mouse anti-rabbit IgG-HRP was incubated during 90 min at room temperature. Finally Western blots were developed by chemiluminescence using Western Blotting Luminol Reagent (sc-2048, Santa Cruz Biotechnology, USA) and analyzed with C-Digit Chemiluminescence Western Blot Scanner (LI-COR Biosciences, USA). β2-Tubulin was used as control.
9
Immunodetection of β-conglutin Proteins
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Purified β-conglutin proteins were separated by SDS-PAGE, using 15 μg of each complete (β5 and β7) and truncated (tβ5 and tβ7) form. After electrophoresis, the proteins were visualized in gel with Coomassie Brilliant Blue for transfer to a PVDF membrane. The membranes were blocked for 2 h in 5% (w/v) non-fat dry milk in Tris-buffered saline (TBS) buffer, pH 7.4. Immunodetection of β-conglutin proteins was carried out by incubation with a primary polyclonal antiserum developed in this study (see above), diluted 1:1000 in TBS buffer containing blocking solution and 0.5% Tween-20. A secondary antibody HRP-conjugated was diluted to 1:3500 in TBS buffer and 0.5% Tween-20 for 2 h, followed by three washing steps of 15 min each with TBS containing 0.5% Tween-20. Protein bands were visualized using a chemiluminescence kit (Bio-Rad) according to the manufacturer’s instructions and the LI-COR C-DiGit Chemiluminescence Western Blot Scanner.
10
Quantifying UCP1 Expression in Mouse Adipose Tissues
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For UCP1 western blotting, 5–20 mg of iBAT, pBAT, ingBAT, pvBAT and gWAT from postnatal day 35 female mice was lysed in T-PER lysis buffer. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with the UCP1 antibody used for immunohistochemistry (1:5000) followed by goat anti-rabbit horseradish peroxidase conjugate secondary antibody (BioRad) and incubation in chemiluminescent reagents. Signal was then captured using a LI-COR C-DiGit Chemiluminescence Western Blot Scanner. Actin was used as a loading control, and was visualized using antibody from Santa Cruz Biotechnology at a dilution of 1:2000. Comparative intensity of UCP1 bands was measured by densitometry using NIH Image J software.
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