After treatments, the cells were washed, and lysed in RIPA lysis buffer (P0013, Beyotime, China) containing phosphatase and protease inhibitors. Heart tissue was homogenized in a lysis buffer (P0013B, Beyotime, China) containing phosphatase and protease inhibitors. The protein samples were resolved and separated by SDS-PAGE, and then transferred onto poly-vinylidene difluoride (PVDF) membranes (Millipore). The membranes were incubated in a blocking buffer, followed by incubation with primary antibodies against specific proteins overnight: Akt (#9272, Cell Signaling Technology [CST], USA), phospho-Akt (serine473, #9271, CST, USA), p62/SQSTM1 (#5114, CST, USA), LC3 (#4108, CST, USA), and GAPDH (sc-25778, Santa Cruz Biotechnology, USA). The primary antibodies were diluted 1:1000 in 5% fat-free milk in TBS buffer overnight. The membranes were washed and incubated with Peroxidase-AffiniPure Goat Anti-Rabbit IgG (111-035-114, Jackson ImmunoResearch). The target-expressed protein-antibody complex was visualized by the PierceTM ECL Western blotting Substrate (#32106, Thermo Scientific). Images with adequate exposure were acquired for densitometry by using the Multi-Gauge software V3.0 from Fujifilm.
Multi gauge software v3
Manufactured by Fujifilm
Sourced in Japan
Multi Gauge software V3.0 is a data analysis software developed by Fujifilm. It is designed to process and analyze data from various measurement instruments. The software provides tools for data visualization, statistical analysis, and report generation.
Automatically generated - may contain errors
Lab products found in correlation
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (3 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Peroxidase affinipure goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Perfecthyb plus hybridization buffer, by Merck Group (1 mentions)
Fuji typhoon fla 7000, by GE Healthcare (1 mentions)
Hybond, by Cytiva (1 mentions)
Enhanced chemiluminescence reagent, by GE Healthcare (1 mentions)
Horseradish peroxidase conjugated goat anti rabbitigg antibody, by Vector Laboratories (1 mentions)
Horseradishperoxidase conjugated horse anti mouse igg antibody, by Vector Laboratories (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
β actin ac 15, by Merck Group (1 mentions)
Perk c33e10, by Cell Signaling Technology (1 mentions)
Atf 4 d4b8, by Cell Signaling Technology (1 mentions)
Peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Fla 7000 phosphorimager, by GE Healthcare (1 mentions)
23 protocols using multi gauge software v3
1
Western Blot Analysis of Cell Signaling
2
Northern blot analysis of RNA
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RNA samples were separated on 4% acrylamide gels containing 7 M urea in 0.5× TBE buffer and transferred to a Hybond N+ membrane by electrotransfer in 0.5× TBE buffer. After transfer, membranes were stained with 0.03% methylene blue in 0.3 M NaAc pH 5.3 for 5 min at RT, scanned, and then destained with water. RNA was immobilized on membranes by 254 nm UV light using a UVP CL-1000 crosslinker. Radioactive probes were labeled with an α32P (dATP) with a DECAprime II DNA Labeling Kit (Thermo Fisher Scientific; AM1455). To obtain templates for probes labeling, PCR on cDNA from B cells activated with LPS and IL-4 for 7 days was conducted using primers listed in Supplementary Table 5 . Alternatively, an oligo probe for U6 as a loading control was labeled with a γ32P (ATP) with T4 PNK (NEB).
Membranes were pre-hybridized in PerfectHyb Plus Hybridization Buffer (Sigma, H7033) for 1 h 65 °C and incubated with radioactive probes in PerfectHyb Plus Hybridization Buffer overnight 65 °C (in case of oligo probe—at 37.5 °C). Then membranes were washed in 2× SSC with 0.1% SDS for 20 min, 0.5× SSC with 0.1% SDS for 20 min and 0.1× SSC with 0.1% SDS for 20 min, scanned with Fuji Typhoon FLA 7000 (GE Healthcare Life Sciences), and analyzed with Multi Gauge software V3.0 (Fujifilm).
Membranes were pre-hybridized in PerfectHyb Plus Hybridization Buffer (Sigma, H7033) for 1 h 65 °C and incubated with radioactive probes in PerfectHyb Plus Hybridization Buffer overnight 65 °C (in case of oligo probe—at 37.5 °C). Then membranes were washed in 2× SSC with 0.1% SDS for 20 min, 0.5× SSC with 0.1% SDS for 20 min and 0.1× SSC with 0.1% SDS for 20 min, scanned with Fuji Typhoon FLA 7000 (GE Healthcare Life Sciences), and analyzed with Multi Gauge software V3.0 (Fujifilm).
3
Western Blotting Analysis of ER Stress Markers
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Tissue samples were homogenized in 20 mM ice-cold Tris-HCl (pH 7.4)
containing 1% protease and phosphatase inhibitors. The homogenates
were centrifuged for 15 minutes at 18,000 × g at 4°C and the resulting
supernatant fractions were used for western blotting. The protein
samples (20 μg/lane) were separated and transferred to hydrophobic
polyvinylidene difluoride membranes. The membranes were blocked and
incubated overnight at 4°C with monoclonal rabbit anti-GRP78,
anti-phospho-NF-κB antibody (1:1,000), monoclonal mouse anti-CHOP,
anti-NF-κB antibody (1:1,000), or monoclonal rabbit anti-IκBα (NF-κ
inhibitor, alpha) antibody (1:1000; Abcam, Cambridge, UK) in 5%
non-fat milk. The next day, the membranes were washed with
phosphate-buffered saline + Tween, and the primary antibody was
detected using a horseradish peroxidase-conjugated goat anti-rabbit
IgG antibody (1:20,000; Vector, Burlingame, CA, USA) or horseradish
peroxidase-conjugated horse anti-mouse IgG antibody (1:5000) in
phosphate-buffered saline for 60 minutes at 25°C. The blots were
developed using an enhanced chemiluminescence reagent (GE Healthcare,
Inc., Piscataway, NJ, USA). The blots were then visualized with a
LAS-3000 Plus lumino-imaging analyzer (Fuji Photo Film Company,
Kanagawa, Japan) and quantified using Multi Gauge software v3.0
(Fujifilm, Tokyo, Japan).
containing 1% protease and phosphatase inhibitors. The homogenates
were centrifuged for 15 minutes at 18,000 × g at 4°C and the resulting
supernatant fractions were used for western blotting. The protein
samples (20 μg/lane) were separated and transferred to hydrophobic
polyvinylidene difluoride membranes. The membranes were blocked and
incubated overnight at 4°C with monoclonal rabbit anti-GRP78,
anti-phospho-NF-κB antibody (1:1,000), monoclonal mouse anti-CHOP,
anti-NF-κB antibody (1:1,000), or monoclonal rabbit anti-IκBα (NF-κ
inhibitor, alpha) antibody (1:1000; Abcam, Cambridge, UK) in 5%
non-fat milk. The next day, the membranes were washed with
phosphate-buffered saline + Tween, and the primary antibody was
detected using a horseradish peroxidase-conjugated goat anti-rabbit
IgG antibody (1:20,000; Vector, Burlingame, CA, USA) or horseradish
peroxidase-conjugated horse anti-mouse IgG antibody (1:5000) in
phosphate-buffered saline for 60 minutes at 25°C. The blots were
developed using an enhanced chemiluminescence reagent (GE Healthcare,
Inc., Piscataway, NJ, USA). The blots were then visualized with a
LAS-3000 Plus lumino-imaging analyzer (Fuji Photo Film Company,
Kanagawa, Japan) and quantified using Multi Gauge software v3.0
(Fujifilm, Tokyo, Japan).
4
Western Blot Experimental Protocol
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For Western blots, cultured cells were lysed in Laemmli SDS sample buffer directly, and brain homogenates were diluted in 2 × Laemmli SDS sample buffer at 1:1 ratio, followed by heating at 95 °C for 5 minutes. Samples were first resolved in 10% SDS-PAGE, and the proteins in the gels were transferred onto Immobilon membrance (Milipore), followed by incubation with primary antibody, washing and then with HRP-conjugated secondary antibodies. The protein-antibody complex were visualized by the Pierce ECL Western Blotting Substrate (Thermo scientific) and exposed to Kodak medical X-ray film (Kodak, USA). Specific immunostaining was quantified by using the Multi Gauge software V3.0 from Fuji Film. Each blot was exposed for 3–4 different periods of time, and only those with adequate exposure were used for quantification.
5
Western Blot Analysis of Stress Signaling
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Whole-cell protein extracts were obtained by lysing cells with Laemmli sample buffer. Protein concentration was measured with a Micro BCA Protein Assay Reagent kit (Pierce, Rockford, IL, USA). Protein extracts (20–40 µg) were subjected to reducing conditions, loaded onto a polyacrylamide gel, and then transferred to Immobilon-P membranes from Millipore (Billerica, MA, USA). One hour after blocking with 5% (w/v) non-fatty milk in Tris-buffered saline solution with Tween® 20, membranes were incubated with the following specific primary antibodies: β-Actin (AC-15, Sigma-Aldrich), ATF4 (D4B8, Cell Signaling), CHOP (2895, Cell Signaling), NOXA (114C307, Abcam), HRI (MBS2538144, MyBioSource), PKR (3072S, Cell Signaling), PERK (C33E10, Cell Signaling), p-PERK (Thr982) (PA5-102853, Invitrogen), and GCN2 (3302S, Cell Signaling). Antibody binding was detected using a secondary antibody conjugated to horseradish peroxidase, and an enhanced chemiluminescence detection system (Amersham, Little Chalfont, UK). Raw quantification of band intensities was performed using Multi Gauge software V3.0 (Fujifilm Corporation).
6
Western Blot Analysis of Hippocampal and Cortical Proteins
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Ipsilateral and contralateral hippocampi and cortices were dissected and homogenized in cold buffer consisting of 50 mM Tris-HCl, pH 7.4, 2.0 mM EDTA, 2.0 mM EGTA, 10 mM β-mercaptoethanol, 150 mM NaCl, 1.0 mM Na3VO4, 50 mM NaF, 10 μg/ml aprotinin, leupeptin and pepstatin, and 0.5 mM AEBSF. The homogenates were boiled in 1× Laemmli buffer (125 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 2.5% β-mercaptoethanol, 0.004% bromophenol blue) for 5 min. Protein concentration was quantified by using A660 Protein Assay kit (Pierce, Rockford, IL, USA). The same amount of brain homogenate proteins was separated by SDS-PAGE and electrically blotted onto polyvinylidene fluoride membrane (PVDF, Millipore). After blocking with 5% milk in Tris-HCl buffered saline (TBS), the membrane was incubated with primary antibodies (Table 1 ) and followed by the species-matched peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA). The blots were then developed by using ECL kit (Thermo Fisher Scientific) and exposed to HyBlot CLr autoradiography film (Denville Scientific, Inc., Holliston, MA, USA). Immunoblotting image was quantified by using the Multi Gauge software V3.0 from Fuji Film (Minato, Tokyo, Japan).
7
Stability of hPGI Dimer against SDS
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The stability of the dimeric structure of hPGI against SDS was analyzed by blue native PAGE, which has been used to characterize the oligomeric state of protein complexes [24] (link). hPGI (10 μg) was mixed with the indicated amounts of SDS and GTP in protein loading buffer, and the protein mixture was incubated at 25 °C for 15 min prior to electrophoresis. The monomer fraction shown on the gel was estimated as pixels with Multi-Gauge software V3.0 (Fujifilm, Tokyo, Japan).
8
Enzymatic Cleavage of Phosphorylated RNA
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Phosphorylated RNA (1.0 µM), including trace amounts of radiolabeled substrate, was subjected to cleavage by either 5.0 nM or 1.0 µM purified Orn at room temperature. These reactions were in the presence of 10 mM Tris, pH 8.0, 100 mM NaCl, and 5 mM MgCl2. At the appropriate times, aliquots of the reaction were removed and quenched in the presence of 150 mM EDTA on ice and heat inactivated at 95°C for 5 min. Activity of whole cell lysates against 32P-labeled oligoribonucleotide substrates was performed at room temperature in reaction buffer (10 mM Tris, pH 8, 100 mM NaCl, and 5 mM MgCl2). At the indicated times, the reaction was stopped by the addition of 0.2 M EDTA and heated at 98°C for 5 min. Samples were separated on denaturing 20% PAGE containing 1x TBE and 4 M urea. The gels were imaged using Fujifilm FLA-7000 phosphorimager (GE) and analyzed for the appearance of truncated 32P-labeled products. The intensity of the radiolabeled nucleotides was quantified using Fujifilm Multi Gauge software v3.0.
9
Protein Extraction and Western Blot Analysis
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Proteins were extracted from RASFs and OASFs using PRO-PREPTM protein extraction buffer (iNtRON Biotechnology Inc., Gyeonggi, Korea). Total protein concentration was measured by Bradford assay using BSA as a standard (Thermo fisher scientific, Waltham, MA, USA). Proteins were separated via 10% or 12% SDS-PAGE and transferred to PVDF membranes (Biorad, Hercules, CA, USA). Membranes were pre-incubated with 5% skim milk in PBS-T (0.1% Tween 20 in PBS) and subsequently incubated with primary antibodies (anti-human GAPDH; Cell signaling, Danvers, MA, USA, anti-human PINK1; Novus bio, Littleton, CO, USA; LC3; Sigma-Aldrich, Burlington, MA, USA, all diluted 1:1000) overnight at 4 °C and incubated with HRP-anti-mouse IgG or HRP-anti-rabbit IgG (1:5000; Cell signaling) for 2 h at room temperature. Membranes were exposed to ECL solution (Thermo fisher scientific, Waltham, MA, USA), and signals were detected using an LAS-4000 luminescent image analyzer (FujiFilm, Tokyo, Japan). Band intensities were quantified using Multi Gauge software V3.0 (Fuji film).
10
Brain Tissue Protein Extraction and Analysis
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Brain tissue was homogenized in cold buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 10 mM β‐ME, 1 mM EDTA, 1 mM Na3VO4, 50 mM NaF, 1.0 mM AEBSF, and 10 μg/ml each of aprotinin, leupeptin, and pepstatin) (1:9 w/v). Brain homogenates were adjusted to 1× Laemmli sample buffer, followed by heating in boiling water‐bath for 5 min. Cultured cells were lysed directly in Laemmli sample buffer and then heated as above. Protein concentration was determined using the Pierce™ 660 nm Protein Assay kit (ThermoFisher Scientific). Samples were subjected to SDS‐PAGE and transferred onto polyvinylidene fluoride membrane (MilliporeSigma, Burlington, MA). The Membrane was subsequently blocked with 5% fat‐free milk in TBS (Tris‐buffered saline) for 30 min, incubated with primary antibody (Table 1 ) diluted in 5% fat‐free milk in TBS containing 0.1% NaN3 overnight, washed with TBST (TBS with 0.05% Tween 20) for three times, and incubated with HRP conjugated secondary antibody for 2 h at RT, washed with TBST, incubated with the enhanced chemiluminescence (ECL) substrate (ThermoFisher Scientific), and exposed to HyBlot CL® autoradiography film (Denville Scientific Inc., Holliston, MA). Specific immunoblotting was quantified by using the Multi Gauge software V3.0 from Fuji Film (Minato, Tokyo, Japan).
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