Kidneys derived from each animal were suspended in RIPA lysis buffer containing a protease inhibitor cocktail (Sigma) and subjected to homogenization as indicated above. Protein denaturation was performed in Laemmli electrophoresis sample buffer (Serva) at 95 °C for 20 min. Protein samples (30 μg/lane) were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad). The membranes were incubated for 12 h (4 °C) with primary antibodies against H3K27ac (rabbit polyclonal, C15410174, dilution 1:1000), H3pan (mouse monoclonal, C15200011, dilution 1:1000), Nox1 (rabbit polyclonal, ab131088, concentration 0.5 µg/mL), Nox2 (rabbit polyclonal, PA5-79118, concentration 0.5 µg/mL), Nox4 (rabbit polyclonal, sc-30141, 1:200), or β-actin (mouse monoclonal, sc-47778, 1:500) followed by one-hour exposure (room temperature) to anti-rabbit IgG-HRP (sc-2370, 1:2000) or anti-mouse IgG-HRP (sc-2031, dilution 1:2000) secondary antibodies. Protein bands were detected by chemiluminescence imaging (ImageQuant LAS 4000 system, Fujifilm, Tokyo, Japan). TotalLab TM (Newcastle upon Tyne, UK)-based densitometric analysis of the protein levels was carried out using the expression level of β-actin protein as internal normalization.
Imagequant las 4000 system
Manufactured by Fujifilm
Sourced in Japan, United States
The ImageQuant LAS-4000 system is a multi-functional lab equipment designed for high-performance imaging and quantification of a variety of samples, including Western blots, gels, and membranes. The system utilizes a high-resolution CCD camera and advanced image capture technology to provide accurate and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Nacalai Tesque (5 mentions)
Anti β actin, by BioLegend (5 mentions)
Las 4000, by Cytiva (5 mentions)
Polyvinylidene difluoride membrane, by Merck Group (4 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Goat anti mouse alkaline phosphatase conjugated secondary antibodies, by Thermo Fisher Scientific (3 mentions)
Anti caspase 8, by Medical & Biological Laboratories (3 mentions)
M per, by Thermo Fisher Scientific (3 mentions)
Phospho dna pkcs, by Abcam (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Rad51, by Santa Cruz Biotechnology (2 mentions)
Anti bcl xs l, by BioLegend (2 mentions)
Anti caspase 3, by Cell Signaling Technology (2 mentions)
Anti β actin, by Abcam (2 mentions)
Ecl system, by GE Healthcare (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Dna pkcs, by Thermo Fisher Scientific (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Cdc25c, by Cell Signaling Technology (1 mentions)
27 protocols using imagequant las 4000 system
1
Western Blot Analysis of Oxidative Stress Markers
2
Western Blot Analysis of DNA Repair Proteins
3
Yeast Protein Expression Analysis
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Galactose-induced or non-induced yeast cells were collected by centrifugation (5,000 × g, 5 min, 4°C) from 500 μl of yeast culture and resuspended with 100 μl of water and 100 μl of 0.2 M NaOH, followed by incubation at room temperature for 5 min. Cells were then collected and resuspended with 100 μl of 1 × yeast SDS sample buffer (60 mM Tris-Cl pH 6.8, 4% β-mercaptoethanol, 4% SDS, 0.01% bromophenol blue, and 5% glycerol) and boiled for 5 min. Yeast microsomal proteins were also prepared from galactose-induced cells harboring BrF3′H or the empty vector for immunoblot analysis. Ten microliters of total protein and 20 μg of microsomal protein were separated by 10% SDS-PAGE and electro-transferred onto a polyvinylidene fluoride membrane. After being blocked with skim milk in TBS-T buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 0.1% Tween 20, and 5% skim milk), the membrane was incubated with anti-His antibodies (Santa Cruz Biotechnology, Dallas, TX, United States) diluted 1/5000 at 4°C for 16 h. The membrane was washed with TBS-T buffer, and HRP-conjugated secondary antibodies (Thermo Fisher Scientific) diluted 1/5000 were then applied at 4°C for 2 h. Chemiluminescent signal was detected using West-Q Femto clean ECL solution (GenDEPOT) and the ImageQuant™ LAS 4000 system (Fujifilm, Tokyo, Japan).
4
Quantitative Western Blot Analysis
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Western blots of whole cell lysates were performed in Lämmli buffer at 25 mA, using TRIS–glycine buffer for transfer 3 h at 100 mA at 4 °C. The membranes were stained with Ponceau S for quality control and marker labeling, blocked in 5% milk powder in PBS-Tween and after primary (O/N at 4 °C) and secondary antibody (1 h) incubation and three times washing in between. The blots were developed using the ECL substrate Western Bright Quantum (Advansta) on an ImageQuant LAS-4000 system (Fuji). The analysis was done in Open FiJi with the gel analysis tool. Shortly, the signal was measured as the area under the profile of intensity of each band over the surrounding background. Then, the signal ratio of each band with respect to its corresponding loading control (Actin or GADPH) was calculated. Calculations, plotting, and statistical analysis were done in Excel and GraphPad Prism software 9.0.
5
Western Blot Analysis of Apoptosis Regulators
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Cells were lysed with the M-PER mammalian protein extraction reagent (Thermo Scientific, Rockford, IL) containing a protease inhibitor cocktail (Nacalai Tesque). Equal amounts of protein were resolved on 4–12% gradient or 12% SDS-PAGE gels, followed by transfer to polyvinylidene fluoride membranes. After blocking membranes, blots were incubated with the indicated primary antibodies: anti-Bcl-2 (sc-492; Santa cruz biotechnology [SCB], Dallas, Texas, USA, anti-Bcl-XS/L (#633901; BioLegend, San Diego, CA, USA ), anti-Mcl-1 (sc-819; SCB ), anti-caspase-3 (9668; Cell Signaling Technology [CST], Danvers, MA, USA ), anti-caspase-8 (M032-3; Medical and Biological Laboratories, Nagoya, Japan), anti-caspase-9 (9508; CST), anti-β-actin (BioLegend ) or anti-α-tubulin (SCB). After washing, room temperature incubation of membranes for 30 min with either goat anti-rabbit or goat anti-mouse alkaline phosphatase-conjugated secondary antibodies (Invitrogen) were used to detect the primary antibodies. Protein bands were visualized using an ImageQuant LAS-4000 system (FujiFilm, Tokyo, Japan).
6
Western Blot Protein Analysis Protocol
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The cells were harvested and lysed with RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) and the resulting protein samples were quantified using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Equal amounts of protein extracts were denatured by boiling at 100°C for 5 min in Laemmli sample buffer (Bio-Rad, Hercules. CA, USA). The proteins were separated by 8-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk in Tris-buffered saline with Tween 20 buffer (TBS-T) (10 mM Tris, 150 mM NaCl, pH 7.5, and 0.1% Tween 20) for 1 h at room temperature, and incubated with primary antibodies (diluted 1:1,000) overnight at 4°C. The membranes were then washed three times for 10 min each with TBS-T buffer and incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies. The membranes were then washed three times for 10 min each with TBS-T buffer, and incubated with Super-signal pico-chemiluminescent substrate or dura-luminol substrate (Thermo Fisher Scientific) according to the manufacturer’s instructions, and visualized using an ImageQuant LAS 4000 system (Fujifilm Life Science, Tokyo, Japan).
7
Western Blot Analysis of Synaptic Proteins
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The Western blot analyses were carried out as previously described26 (link). PVDF membranes were incubated with the following primary antibodies: anti-beta actin (1:5000), anti-DBN (drebrin) (1:300), anti-MUNC13-4 (1:800), anti-RAB3A (1:200), anti-SV2A (1:300), anti-SV2C (1:300), anti-SYNG1 (synaptogyrin 1) (1:600), anti-SYT1 (synaptotagmin 1) (1:250) (all from Abcam), anti-NSF (1:750), anti-SNAP25 (1:400) (Cell Signaling Technology), anti-RAB3C (1:1000) (Synaptic Systems), anti-RPH3A (rabphilin 3A) (1:1000) (Santa Cruz), anti-SV2B (1:150), anti-SYP (synaptophysin) (1:200) (Sigma-Aldrich) or anti-SYN1 (synapsin 1) (1:200) (Millipore). Anti-rabbit IgG-HRP (Santa Cruz) or anti-mouse IgG-HRP (Cell Signaling Technology) were used as secondary antibodies. The target proteins were detected with the Novex ECL HRP Chemiluminescent Reagent Kit (Invitrogen) and an ImageQuant LAS 4000 system (FUJI FILM).
8
Protein Expression Analysis by Western Blot
9
Western Blot Analysis of Breast Cancer Cells
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Harvested human breast cancer cells were washed with ice-cold phosphate-buffered saline (PBS) and total protein was extracted using radioimmunoprecipitation assay buffer (Sigma-Aldrich) containing a protease inhibitor cocktail (Sigma-Aldrich). Concentrations of extracted proteins were measured by Bradford assay [15 (link)]. Each 40 µg of protein from cell lysate was separated by 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto nitrocellulose membrane (Thermo Fisher Scientific). Blots were blocked with 5% nonfat milk in trisbuffered saline containing 0.1% Tween 20 at room temperature for 2 hours. These membranes were subsequently incubated at 4℃ overnight with corresponding primary antibodies. Blots were then incubated with horseradish peroxidase-conjugated immunoglobulin G secondary antibody at room temperature for 1h. Following incubation with enhanced chemiluminescence solution (RPN2106, GE Healthcare, Little Chalfont, UK), protein bands were detected by ImageQuant LAS-4000 system (Fujifilm, Tokyo, Japan). Signal intensity was normalized to that of β-actin as loading control.
10
Quantitative Western Blot Analysis of Collagen III
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Cells were lysed in 2X Laemmli buffer containing proteases inhibitor cocktail (Merck KGaA, Darmstadt, Germany), and protein concentration was determined using the Amido Black assay. A total of 40 μg of protein was loaded and resolved on 4–15% of precast polyacrylamide gel, followed by a transfer on 0.45 μm nitrocellulose membrane and Ponceau S reversible staining. After blocking 3 h in 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T), the membranes were incubated with anti-collagen III antibody (Abcam, Cambridge, UK, ab7778, 1 μg/ml) overnight at 4°C. Membranes were rinsed in TBS and further incubated with HRP-conjugated secondary antibody (R&D Systems, Canada, HAF008) for 1 h. Protein bands were imaged after enhancement with a chemiluminescence agent (Thermo Fisher Scientific, MA, USA) using an ImageQuant LAS4000 system (Fujifilm, Minato, Japan). The protein expression was quantified by densitometry with TotalLab software and normalized to total protein as quantified by Ponceau S staining.
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