Biotin-Sph (1 nmol) and MBP-SphR (0.2 nmol) were mixed in 100 μl of binding buffer (20 mM Tris-HCl, pH 7.5 containing 100 mM KCl, 2 mM DTT, 8% glycerol, 6 mM MgCl2, 100 μg/ml BSA, 2 mM EDTA, and 1% NP-40) and incubated at room temperature for 30 min. Dynabeads MyOne Streptavidin T1 (Thermo Fisher Scientific) was added to the mixture and incubated at room temperature for 30 min. Beads were separated using a magnet, the supernatant containing non-bound MBP-SphR was removed, and the beads were washed three times with 0.1% NP-40 in TBS using magnetic separation. The beads obtained were suspended in 45 μl of SDS sample buffer. After boiling at 100 °C for 5 min, the sample (20 μl) was subjected to SDS-PAGE and stained by EzStain Aqua (ATTO). The stained gel was scanned and band intensities were analyzed using Multi Gauge software version 3.1 (FUJIFILM Corporation, Tokyo, Japan).
Multi gauge software version 3
Manufactured by Fujifilm
Sourced in Japan
Multi Gauge software version 3.0 is a measurement and analysis software tool designed for Fujifilm's lab equipment. The software provides core functions for capturing, processing, and analyzing measurement data from various laboratory instruments.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride membrane, by Merck Group (4 mentions)
Enhanced chemiluminescence western blot analysis kit, by Thermo Fisher Scientific (3 mentions)
Las 4000 mini, by Fujifilm (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Immobilon p polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Las 3000 mini imaging analyzer, by Fujifilm (2 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Las 4000 luminescent image analyzer, by Fujifilm (2 mentions)
Horseradish peroxidase conjugated anti rabbit igg 7074, by Cell Signaling Technology (2 mentions)
Donkey anti goat igg a50 101p, by Fortis Life Sciences (2 mentions)
Goat anti mouse igg sc 2031, by Santa Cruz Biotechnology (2 mentions)
Dynabeads myone streptavidin t1, by Thermo Fisher Scientific (1 mentions)
Ecl reagent, by GE Healthcare (1 mentions)
Las 4000 imaging system, by Fujifilm (1 mentions)
Horseradish peroxidase, by GE Healthcare (1 mentions)
Dctm protein assay, by Bio-Rad (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Las 1000, by Fujifilm (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
28 protocols using multi gauge software version 3
1
Biotin-Sph and MBP-SphR Binding Assay
2
Liver Protein Extraction and Western Blot Analysis
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The frozen powdered liver tissue samples were homogenized in an ice-cold buffer containing 25 mM HEPES, 25 mM benzamidine, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1% triton X-100, 4 mM EDTA, 5 μL/mL of phosphatase inhibitor, and 5 μL/mL of protease inhibitor, sonicated for 1 minute, and centrifuged at 14,000 ×g, for 30 minutes at 4°C. Total protein concentrations were quantified using a BCA kit (Pierce, Rockford, IL, USA). The extracted proteins were separated on 8–13.5% SDS polyacrylamide gels and transferred to nitrocellulose membranes. Primary antibodies against total and phosphorylated AMPK (Cell Signaling Technology, CST, Denver, MA, USA; 1 : 5000), total and phosphorylated mTOR (CST; 1 : 1000), LC3B (CST; 1 : 1000), caspase-3 (CST; 1 : 1000), total and phosphorylated ERK-1 (CST; 1 : 5000), total and phosphorylated Akt (CST; 1 : 5000), and total and phosphorylated PTEN (CST; 1 : 1000) were applied overnight at 4°C. The membranes were then developed using horseradish peroxidase-conjugated anti-rabbit IgG (CST; 1 : 5000) followed by detection with ECL reagent (GE healthcare, Wauwatosa, WI, USA). The immunoreactive protein bands were quantified using Multi Gauge software version 3.1 (Fujifilm, Tokyo, Japan).
3
Western Blot Analysis of Cell Signaling
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The total cell lysate was harvested, as previously described [17 (link)]. Briefly, 30 μg of extracted protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto polyvinylidene fluoride (PVDF) membranes, and then probed with primary and secondary antibodies according to the manufacturers’ instructions. The primary antibodies used in the immunoblot included: p-STAT5B (1: 1000), STAT5B (1: 1000), cyclin D1 (1: 1000), IGF-1 (1: 1000), Bcl-2 (1: 1000), Bcl-xL (1: 1000), cPARP (1: 1000), p-AKT (1: 1000), AKT (1: 1000), β-actin (1: 2000), and secondary horseradish peroxidase (HRP)-conjugated antibodies (1: 5000). Blots were recorded using a LAS-4000 imaging system (Fujifilm, Minato, Tokyo, Japan) and by chemiluminescence (Thermofisher Scientific, Waltham, MA, USA), and quantified using Multi Gauge software version 3.1 (Fujifilm, Minato, Tokyo, Japan).
4
Signaling Pathways Activation in dHL-60 Cells
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dHL‐60 cells (1 × 106 cells) were stimulated with 3nM C5a for 15, 30, and 120 min. dHL‐60 cells were lysed with RIPA buffer containing protease and phosphatase inhibitors. Next, the protein concentration was measured using DCTM Protein Assay (Bio‐Rad, Hercules, CA). Then, 10 µg of each protein was applied to 10% polyacrylamide gel, and the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (GE Healthcare UK Ltd.). Membranes were reacted with anti‐phospho (p)‐NF‐κB p65, NF‐κB p65, p‐ERK1/2, ERK1/2, p‐p38 MAPK, and p38 MAPK (Santa Cruz Biotechnology) following anti‐mouse or anti‐rabbit secondary antibody conjugated with horseradish peroxidase (GE Healthcare UK Ltd.). Immunoreactive proteins were detected with a luminol‐based enhanced chemiluminescence kit (Thermo Scientific). The signals were detected by LAS‐1000 (Fujifilm) and measured using Multi Gauge Software version 3.0 (Fujifilm). The relative expression of phosphorylated proteins was calculated by using 1.00 for the expression level of total proteins.
5
Western Blot Analysis of Protein Samples
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Cells were lysed with lysis buffer containing 1% Triton X-100, and the resulting samples separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore Corporation, Billerica, MA, USA). Immunoblot analysis was performed and visualized using Super-Signal West Pico Chemiluminescent substrate (Pierce Chemical Co., Rockford, IL, USA) or Immobilon Western Chemiluminescent HRP substrate (Millipore Corporation, Billerica, MA, USA). Signal intensities were analyzed using an LAS-3000 mini imaging analyzer and Multi Gauge software, version 3.0 (Fujifilm, Tokyo, Japan).
6
Extraction and Characterization of Extracellular Vesicles
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When MeT-5A cells grown in 10-cm dishes containing maintenance medium were 50% confluent, they were washed twice with PBS, and added with FBSpas50-containing medium. After 48 h, cultured medium or fresh medium containing FBSpas50 (for the negative control) was collected and passed sequentially through 220 and 50 nm syringe filters. Proteins of EVcap50/220 were eluted with 2 ml CHAPS lysis buffer (Dojindo) containing 1 mM PMSF. Each eluted solution was concentrated using Amicon Ultra 10 kDa (Merck Millipore) until its volume was less than 30 µl. All samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to polyvinylidene difluoride (PVDF) membranes. The membrane was immunoblotted first with anti-EpCAM antibody (EBA-1, dilution 1:1,000, Santa Cruz Biotechnology, Inc.), CD63 antibody (TS63, dilution 1:1,000, Thermo Fisher Scientific, Inc.), CD9 antibody (TS9, dilution 1:1,000, Thermo Fisher Scientific, Inc.), CD81 antibody (M38, dilution 1:1,000, Thermo Fisher Scientific, Inc.). Secondary antibodies conjugated with horseradish peroxidase were used for detection. Signal intensity was obtained using LAS 4000 Mini and Multi Gauge software version 3.0 (Fujifilm), as previously described (17 (link)).
7
Protein Expression Analysis of hESCs
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Total protein lysates were extracted from HESCs by direct lysis in SDS sample buffer heated to 75 °C. Proteins resolved by SDS-PAGE were separated in a polyacrylamide gel (Bio-rad laboratories, Hercules, USA) and transferred to Immobilon-P membrane (Merck, Darmstadt, Germany) and probed with antibodies raised against SIRT1, 1:500 (Abcam) CRABP2, 1:5000 (Sigma-Aldrich); FABP5, 1:1000 (Abcam); RARα, 1:5000 (Abcam); PPARβ/δ, 1:1000 (Abcam); p53, 1:1000 (Cell Signaling Technology, Danvers, USA), and β-actin, 1:5000 (Sigma-Aldrich). After incubation with peroxidase-conjugated secondary antibodies, (Jackson Immunoresearch Laboratories, West Grobe, USA), detection of chemiluminescence was visualized with Pierce Western Blotting Substrate Plus (Thermo Fisher Scientific, Rockford, USA) or Super Signal West Dura Extended Duration Substrate (Thermo Fisher Scientific) and normalized to β-actin. Western blots were quantified by densitometry using Multi Gauge software Version3.0 (Fuji Photo Film, Tokyo, Japan).
8
Immunoblotting Analysis of proMMP-9 from THP-1 and Sf9 Cells
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Purified proMMP-9 from THP-1 cells and recombinant full length human proMMP-9 from Sf9 cells with and without 0.1 M DTT were electrophoresed on SDS-polyacrylamide gel (NuPAGE Novex 4–12% Bis-Tris gels) and electroblotted to a polyvinyl difluoride membrane. After blockage of non-specific binding sites with non-fat milk in TBS-T (150 mM NaCl, 0.25% Tween-20, 20 mM Tris-HCL, pH 7.4), blots were incubated for 1 h at room temperature with primary rabbit polyclonal antibody against human MMP-9. After washing, the blots were incubated for 1 h at room temperature with an HRP-conjugated goat anti-rabbit secondary antibody. The Blots were thereafter washed with TBS-T 3 x 5 min before visualization using Western Blotting Luminol reagent. The intensity of immunoblot bands was measured using a Luminescent Image Analyzer LAS-3000 with MultiGauge software version 3.0 (Fujifilm, Tokyo, Japan).
9
AtPol λ Activity Assay
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Reactions (10 μl) contained 50 mM Tris HCl pH 7.5, 1 mM DTT, 0.2 mg/ml BSA, 2% glycerol, 10 mM MgCl2, 100 nM DNA substrate, the indicated amount of AtPol λ and dCTP, dNTPs (deoxynucleotides) or ddNTPs (dideoxynucleotides). When indicated, AtPol λ was pre-incubated with pre-immune serum or anti-AtPol λ antibody for one hour at 4 °C. After incubation at 37 °C for the indicated times, reactions were stopped by adding 20 mM EDTA, 0.6% SDS and 0.5 mg/ml proteinase K, and mixtures were incubated at 37 °C for 30 min. DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and ethanol precipitated at -20 °C in the presence of 0.3 mM NaCl and 16 mg/ml glycogen. Samples were resuspended in 10 µl of 90% formamide and heated at 95 °C for 5 min. Reaction products were separated in a 12% denaturing polyacrylamide gel containing 7 M urea. Labelled DNA was visualized using FLA-5100 imager (Fujifilm) and analyzed using Multi Gauge software version 3.0 (Fujifilm).
10
Western Blot Protein Detection Protocol
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Equal amounts of protein (30 µg) were separated using 7–12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (EMD Millipore). The membranes were blocked by incubation with 1% bovine serum albumin (BSA) in TBS-T (10 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween-20) at room temperature for 90 min, and then incubated with specific primary antibodies at room temperature for 3 h (Table I ). The membranes were washed three times with TBS-T and incubated for 90 min at room temperature with the appropriate horseradish peroxidase-conjugated secondary anti-rabbit IgG (cat. no. 7074S; Cell Signaling Technology, Inc.), anti-mouse IgG (cat. no. 7076S; Cell Signaling Technology, Inc.), or donkey anti-goat IgG antibodies (cat. no. A50-101P; Bethyl Laboratories, Inc.), diluted at 1:10,000-1:20,000 in TBS-T containing 1% BSA. Signals were detected using an enhanced chemiluminescence western blot analysis kit (Thermo Fisher Scientific, Inc.). Experiments were performed in triplicate, and densitometry analysis was performed using Multi-Gauge software version 3.0 (Fujifilm Life Sciences). GAPDH, β-actin, or lamin B1 were used as internal controls for equal protein loading in total cell, cytoplasmic or nuclear extracts, respectively (18 (link)).
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