Protein lysates of all cells were extracted with RIPA buffer (Wako Pure Chemical Industries Ltd, Osaka, Japan) including Complete Protease Inhibitor Cocktail and PhosSTOP Phosphatase Inhibitor Cocktail (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. Ten μg of the protein lysates were loaded onto Mini-PROTEIN TGXTM gel (Bio-Rad Laboratories). After electrophoresis, proteins were transferred to PVDF membranes using the Trans-Blot TurboTM Transfer System Transfer Pack (Bio-Rad Laboratories). Subsequently, the membranes were blocked for 1 h in 4% BSA in TBS with 0.5% Tween-20 (PBS-T) and incubated overnight at 4 °C with primary antibody in TBS-T with 4% BSA. Antibodies concentrations in this study were 1/2500 for rabbit anti-HtrA2 (Cell Signaling Technology) and 1/5000 for HRP-conjugated β-actin (Cell Signaling Technology). After washing three times with TBS-T, membranes were incubated using HRP-conjugated anti-rabbit secondary antibody (Cell Signaling Technology) for 1 h at room temperature. After washing three times with TBS-T, they were visualised using the ECL detection system (GE Healthcare UK Ltd, England, UK). Pictures were taken using a LAS-3000 (Fujifilm, Minato, Tokyo). Protein expression was determined densitometrically and normalised against β-actin expression using the Multi Gauge version 3.1 software (Fujifilm).
Multi gauge version 3
Manufactured by Fujifilm
Sourced in Japan, United States
Multi Gauge version 3.0 software is a tool for analyzing and measuring data from various sources. It provides a user-friendly interface for visualizing and processing information. The software is designed to be a versatile and efficient solution for data analysis tasks.
Automatically generated - may contain errors
Lab products found in correlation
Las 3000, by Fujifilm (9 mentions)
Las 4000, by Cytiva (6 mentions)
Las 4000, by Fujifilm (4 mentions)
β actin, by Merck Group (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Pro prep protein extraction solution, by iNtRON Biotechnology (3 mentions)
Ripa buffer, by Fujifilm (2 mentions)
Ecl detection system, by GE Healthcare (2 mentions)
Cox 2, by Cell Signaling Technology (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Las 4000 mini, by GE Healthcare (2 mentions)
Typhoon fla 9500, by GE Healthcare (2 mentions)
Las 3000 imaging system, by Fujifilm (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Las 3000 mini, by Fujifilm (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (2 mentions)
Hrp conjugated β actin, by Cell Signaling Technology (1 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (1 mentions)
66 protocols using multi gauge version 3
1
Western Blot Analysis of HtrA2 Protein
2
Quantitative RT-PCR Analysis of Placental HIF and sFlt-1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from placental tissue in RNAlater using the RNeasy® Mini Kit (Qiagen, Hilden, Germany) and RNA concentrations determined using the Nanodrop. RNA (1 μg) was reverse transcribed using SuperScript™III (Invitrogen, Waltham, MA, USA). PCR primers and conditions are outlined in Table 2 . RT-PCR products were separated by electrophoresis on 1% (w/v) agarose gels or a microchip electrophoresis system (MultiNA; Shimadzu, Kyoto, Japan) as indicated. Relative intensities of bands for HIF-1α, HIF-2α and sFlt-1 were quantitated after normalisation to β-actin using MultiGauge version 3.1 software (Fujifilm, Macquarie Park, NSW, Australia).
3
In Vitro Kinase Assay for GST Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In vitro kinase assay was done, as shown in the former studies [50 ]. Briefly, GST protein expressed in Escherichia coli was lysed with a RIPA buffer (50-mM Tris-HCl pH 7.4, 150-mM NaCl, 0.1% SDS, 0.5% DOC, 1% NP-40) having a complete protease inhibitor cocktail (Roche) and bound to Glutathione Sepharose 4B beads (GE Healthcare). Then, the GST protein was rinsed with a kinase buffer (40-mM Tris-HCl pH 7.4, 20-mM MgCl2, 0.1-mg/ml bovine serum albumin, and 50-μM dithiothreitol) and eluted using a kinase buffer containing 20-mM reduced glutathione. The RC-DC Protein Assay (Bio-Rad) was applied to measure the GST protein levels.
For the kinase assay, 16.7-ng/μl substrate candidate protein, 0.33-ng/μl ARK5 kinase, 33.3-nCi/μl (11 nM) [γ32-P] ATP, and 50-µM non-radiolabeled ATP were mixed in a kinase buffer (reaction volume of 30 μl) and stored for 30 min at 30 °C. The end of the reaction was marked by blending with 4× Laemmli sample buffer (40% glycerol, 270-mM Tris-HCl pH 6.8, 8% SDS, 20% 2-mercaptoethanol, 0.006% BPB). Then, the samples were detached by SDS-PAGE. In the end, the autoradiography image was obtained with BAS-5000 (GE Healthcare), Image Reader BAS-5000 Version 1.8 (Fujifilm), and Multi Gauge Version 3.1 (Fujifilm) software.
For the kinase assay, 16.7-ng/μl substrate candidate protein, 0.33-ng/μl ARK5 kinase, 33.3-nCi/μl (11 nM) [γ32-P] ATP, and 50-µM non-radiolabeled ATP were mixed in a kinase buffer (reaction volume of 30 μl) and stored for 30 min at 30 °C. The end of the reaction was marked by blending with 4× Laemmli sample buffer (40% glycerol, 270-mM Tris-HCl pH 6.8, 8% SDS, 20% 2-mercaptoethanol, 0.006% BPB). Then, the samples were detached by SDS-PAGE. In the end, the autoradiography image was obtained with BAS-5000 (GE Healthcare), Image Reader BAS-5000 Version 1.8 (Fujifilm), and Multi Gauge Version 3.1 (Fujifilm) software.
4
Axon Morphometric Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Densitometry analysis was performed with Multi Gauge, version 3.1 (Fujifilm). Morphometric analysis of axons was performed as described previously [7] (link). A cell was considered to have an axon if the length of one neurite was at least twice as long as any other process and was more than twice the diameter of the cell body, as described previously [30] (link). The total length of axon and the number of axon tips were measured. Only processes longer than 5 µm in length were included in the analysis [31] (link). Statistical significance was established at p < 0.05 by one-way analysis of variance (ANOVA) with post hoc test (Dunnett) or Student's t-test using KaleidaGraph (4.5.2; Synergy Software).
5
Immunoblot Analysis of Inflammatory Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted using lysis buffer and quantified using the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA), as described previously (Hong et al. 2020 ). The samples were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After blocking, the membranes were immunoblotted with primary antibodies COX-1(Abcam, 1:1000), COX-2 (LSBio, 1:1000), NF-κB (Cell Signaling Technology, 1:1000), TNF-α (Abcam, 0.2 μg/ml), or IL-6 (Thermo Fisher, 1:1000), and β-actin (Cell Signaling Technology, 1:1000) at 4°C overnight. After washing, the membranes were incubated at 37°C for 1 h with the horseradish peroxidase (HRP)-conjugated secondary antibodies (Enzo Life Sciences, 1:4000). An enhanced chemiluminescence reagent (Millipore, Billerica, MA, USA) was added, and the light emission was detected using a luminescent image analyzer (LAS-4000, Fuji Film Co., Japan). The intensity of bands was measured using Multi-Gauge Version 3.1 (Fuji Film Co., Japan). The value of each sample was expressed as the percentage of the control tested protein relative to the internal reference, β-actin.
6
Phospho-ERK Western Blot Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transfected cells were cultured in 6-well plates, and were starved overnight in a serum-free culture medium containing 0.1% bovine serum albumin (BSA). Cells were treated with 5-HT dissolved in serum-free culture medium, and sodium dodecyl sulfate (SDS) sample buffer was directly added to culture wells. After incubating for 20 min at 65°C, samples were sonicated to shear genomic DNA. Proteins were separated by SDS-polyacrylamide gel electrophoresis (10% running gel, 5% stacking gel) and electroblotted onto polyvinylidene difluoride or nitrocellulose membranes. The membranes were incubated for 1 h at 22°C in TBS-Tween 20 (TBS-T) containing 5% nonfat dry milk or 4% BSA, followed by 1 h of incubation with antibody to phospho-ERK (1:1,000 dilution) and 1 h with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000 dilution) in 2% nonfat dry milk. Blots were visualized with chemiluminescent western blotting kit. The same samples were processed to detect ERK. Signals were quantified using Multi Gauge version 3.1 (FUJIFILM Corporation, Tokyo, Japan).
7
Western Blot Analysis of Apoptosis and Inflammation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples (10 to 40 μg) were subjected to SDS-PAGE and transferred to Immobilon P membranes (Millipore Corp., Billerica, MA). After blocking, membranes were probed with primary antibodies against active + pro caspase 3 (1:1000; Abcam), α SMA (1:1000; Abcam), phosphorylated NF-κB-p65 (1:1000; Cell Signaling, MA, USA), total NF-κB-p65 (1:1000; Delta Biolabs), Cytochrome c (1:1000; Santa Cruz Biotechnology, CA, USA), GAPDH (1:2000; Santa Cruz Biotechnology, CA, USA), CD10 (1:1000; R&D System, MN, USA), iNOS (1:1000; Abcam), HO-1 (1:1000; Assay designs, NY, USA), or CYGB (1:3000; our laboratory). Membranes were then incubated with horseradish peroxidase conjugated secondary antibodies at 1:2000 dilutions. Immunoreactive bands were visualized using the electrochemiluminescence detecting reagent (GE Healthcare UK Ltd, Buckinghamshire), and documented with the Fujifilm Image Reader LAS-3000 (Fujifilm, Tokyo, Japan) coupled with image analysis software (Multi Gauge version 3.1; Fujifilm, Tokyo, Japan).
8
Sepsis-induced Liver Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The liver of mice was dissected 1 h and 3 h after sepsis induction. After dissection from hippocampus and liver, tissue was lysis with SDS lysis buffer containing 0.1 mM Na3VO4, and 20 mM NaF. After brief sonication to shear DNA and reduce viscosity, the concentration of protein was determined with the detergent-compatible protein assay reagent (Bio-Rad Laboratories, USA) using bovine serum albumin as the standard. The western blot analysis was performed according to our previous study [38 (link)42 (link)]. The membranes were then exposed to a Luminescent Image Analyzer (LAS-4000, Fuji Film Co., Japan) for the detection of light emission. Specific signals were quantified with the Multi-Gauge Version 3.1 (Fuji Film) and expressed as a percentage of the control.
9
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein lysates were extracted in RIPA buffer® according to the manufacturer's instructions (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Ten μg of cytosolic fractions were and loaded onto Mini-PROTEIN® TGX™ gel (Bio-Rad Laboratories). After electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF) membranes using Trans-Blot® Turbo™ Transfer System Transfer Pack (Bio-Rad Laboratories). Subsequently, the membranes were blocked for 1 hour in 4% bovine serum albumin (BSA) in tris buffered saline (TBS) with 0.5% Tween-20 (TBS-T) and incubated overnight at 4°C in primary antibody in TBS-T with 4% BSA. The following antibodies and concentrations were used: 1/2,500 rabbit LC3A, 1/2,500 rabbit PARP, 1/2,500 rabbit cleaved-PARP, 1/5,000 rabbit β-actin. After 3 washes with TBS-T, membranes were incubated for 1 hour at room temperature using horseradish peroxidase-conjugated anti-rabbit secondary antibody as appropriate. After 3 washes with TBS-T, they were visualized using the ECL detection system (GE Healthcare UK Ltd., England, UK) by a LAS-3000 (Fujifilm, Tokyo, Japan). Protein expression was determined densitometrically and normalized against β-actin expression using Multi Gauge version 3.1 (Fujifilm).
10
Statistical Analysis of Experimental Data
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In all experiments, data was quantitatively analyzed for statistical significance using either a Student's t-test (two-tailed) or a one-way ANOVA followed by a Tukey–Kramer multiple comparisons test. The Student's t-test was applied for comparisons of two groups. GraphPad Prism, Version 5.0 (GraphPad Software, San Diego, CA, USA) software was used and differences were significant when P<0.05. Western blotting data was assessed using MultiGauge version 3.1 (Fuji, Japan) software and converted into ratios of band intensity relative to the controls. Eye size was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The Kaplan–Meier estimator and the log rank test were conducted on the cumulative survival under oxidative stress conditions and lifespan data to determine whether each condition had any effect on the longevity of individuals by using Online Application for the Survival Analysis of Lifespan Assays (http://sbi.postech.ac.kr/oasis ).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!