Exosome samples (2 µg) were reduced by 100 mM dithiothreitol at 95°C for 3 min, subjected to 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and electrophoretically transferred to a polyvinylidene fluoride transfer membrane. The membrane was blocked with Blocking One (Nacalai Tesque, Kyoto, Japan) for 30 min. In order to detect Alix, the membrane was probed with mouse anti-Alix antibody (1:20,000 dilution; BD Biosciences, San Jose, CA, USA) for 1 h at room temperature, and then allowed to react with rabbit anti-mouse IgG antibody conjugated with horseradish peroxidase (1:2,000 dilution; Life Technologies, Grand Island, NY, USA) for 1 h at room temperature. In order to detect Hsp70, the membrane was probed with rabbit anti-Hsp70 antibody (1:1,000 dilution; Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature, and then allowed to react with goat anti-rabbit IgG antibody conjugated with horseradish peroxidase (1:5,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. The membrane was soaked with Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Billerica, MA, USA), and chemiluminescence was detected by a LAS-3000 instrument (FUJIFILM, Tokyo, Japan).
Las 3000 instrument
Manufactured by Fujifilm
Sourced in Japan
The LAS-3000 instrument is a versatile lab equipment designed for capturing and analyzing images. It features a high-resolution camera, advanced imaging software, and a range of accessories to support various imaging applications. The core function of the LAS-3000 is to provide users with a comprehensive solution for capturing, processing, and analyzing images in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Nucleospin rna protein kit, by Macherey-Nagel (2 mentions)
β actin, by Fortis Life Sciences (2 mentions)
Chemiluminescence detection system, by PerkinElmer (2 mentions)
Anti α tubulin, by Merck Group (2 mentions)
Tween 20, by Merck Group (2 mentions)
Rabbit anti hsp70 antibody, by Cell Signaling Technology (1 mentions)
Mouse anti alix antibody, by BD (1 mentions)
Goat anti rabbit igg antibody conjugated with horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Blocking one, by Nacalai Tesque (1 mentions)
B9643, by Merck Group (1 mentions)
C2284, by Merck Group (1 mentions)
Lc3a b, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Protease inhibitor, by Nacalai Tesque (1 mentions)
Specific antibodies, by Cell Signaling Technology (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by GE Healthcare (1 mentions)
Pdvf membrane, by Bio-Rad (1 mentions)
Phosstop, by Roche (1 mentions)
13 protocols using las 3000 instrument
1
Western Blot Analysis of Exosome Proteins
2
Protein Isolation and Western Blot Analysis
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Proteins were isolated from cells and kidney tissue using NucleoSpin RNA/Protein kits (Macherey-Nagel). Protein concentration was calculated using the Bradford assay with bicinchoninic acid (B9643, Sigma) and copper sulfate (C2284, Sigma) solutions. Proteins were separated by 8–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (AE-6667-P, ATTO). The following primary antibodies were used in this study: Atg5 (#12294, Cell Signaling Technology), LC3 A/B (#12741, Cell Signaling Technology), p62 (#5114 and #23214, Cell Signaling Technology), Pten (#9552, Cell Signaling Technology), pDVL (ab124933, Abcam), Dvl2 (#3224, Cell Signaling Technology), and β-actin (A300-491A, Bethyl Laboratories).
Membranes were blocked with 5% skimmed milk in PBST (1 × PBS with 1% Tween-20), incubated with primary antibodies diluted in 1% skimmed milk with PBST overnight at 4°C, washed with PBST, and incubated with secondary antibodies in 2% skimmed milk for 1 h at room temperature. Protein bands were detected using enhanced chemiluminescent reagents (WSE-7120 EzWestLumi plus, ATTO) and band intensity was visualized using an LAS-3000 instrument (Fujifilm).
Membranes were blocked with 5% skimmed milk in PBST (1 × PBS with 1% Tween-20), incubated with primary antibodies diluted in 1% skimmed milk with PBST overnight at 4°C, washed with PBST, and incubated with secondary antibodies in 2% skimmed milk for 1 h at room temperature. Protein bands were detected using enhanced chemiluminescent reagents (WSE-7120 EzWestLumi plus, ATTO) and band intensity was visualized using an LAS-3000 instrument (Fujifilm).
3
Gel Electrophoresis of PCR Products
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After PCR, tubes were centrifuged for 3 min, and 25 μl supernatant was removed, mixed with 10 μl of 200 μg/ml Proteinase K, and incubated at 60°C for 90 min. We subsequently mixed 12 μl samples with 2 μl loading buffer (Nippon Gene), loaded samples onto a 2% agarose gel, performed electrophoresis and detected fluorescein in the gel using an LAS-3000 instrument (Fujifilm). A 4 μl sample of FluoroBand 100 bp Fluorescent DNA ladder (SMOBIO Technology) served as a size marker.
4
Exosome Characterization by Western Blot
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To characterize the exosomes, purified exosomes and cell extracts were lysed in the RIPA lysis buffer (150 mmol/L NaCl, 1% Triton X‐100, 0.5% sodium deoxycholate, 1% SDS, 50 mmol/L Tris (pH = 8)) and a protease inhibitor cocktail (Sigma, USA). After lysis, 15 µg of each sample was re‐suspended in reducing sodium dodecyl sulphate (SDS) loading buffer and incubated for 5 minutes at 95°C. Thereafter the samples were subjected to 12% SDS‐polyacrylamide gel electrophoresis. Following electrotransfer of separated proteins onto a polyvinylidene fluoride (PVDF) transfer membrane and a blocking step, the transferred proteins were immunodetected using the rabbit anti‐human anti‐CD63 and anti‐CD81 primary antibody (Exosomes Antibodies Array & ELISA Kits (EXOAB‐KIT‐1, System Biosciences (SBI), UK, 1:1000) overnight, followed by incubation with appropriate goat anti‐rabbit IgG horseradish peroxidase (HRP)‐coupled secondary antibody (1:20 000) for 1 hour at room temperature. The blot was subsequently developed using a chemiluminescent HRP substrate and chemiluminescence was detected using a LAS3000 instrument (Fujifilm, Japan). The primary antibody was omitted in the control group and rabbit IgG was used as isotype control.
5
Immunoblotting Analysis of Protein Redox Status
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Three-day cultures under low light or 2-d cultures under high light of WT cells were precipitated with 10% (w/v) TCA and washed with ice-cold acetone. For dark conditions, the culture tube was covered with aluminum foil for 3 h before TCA precipitation. The cells were treated with 25 mM DTTred for 10 min at room temperature before TCA precipitation for the DTT treatment condition. Precipitates were then suspended in SDS sample buffer containing 4 mM PEO. After labeling for 40 min at room temperature, protein samples were boiled at 95°C for 5 min and centrifuged at 20,400×g for 10 min. Proteins in the supernatants were then separated by nonreducing SDS-PAGE using 10% (w/v) polyacrylamide gel with 8 M urea in the separating gel and blotted onto polyvinylidene difluoride membranes. Immunoblotting was performed using PRK polyclonal antibodies against recombinant AtPRK. The chemiluminescence of the horseradish peroxidase-conjugated secondary antibody was detected using an LAS 3000 instrument (Fujifilm, Tokyo, Japan).
6
Cisplatin-induced Caspase Activation Assay
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After incubating cells with cisplatin, cells were collected, lysed in a RIPA buffer with protease inhibitors (Nacalai Tesque), and subjected to 10~15% SDS-PAGE. The electrophoresed proteins were transferred onto a polyvinyidene difluoride membrane (Millipore, Billerica, MA) using a semidry blot system (2 mA/cm2 for 0.5 hr in 0.2 M Tris-glycine buffer, pH 8.8). Caspase-3 and -9 activation was analyzed using specific antibodies (Cell Signaling Technology, Beverly, MA). After incubation of the sheets in TBS solution (140 mM NaCl, 50 mM Tris-HCl, pH 7.2) containing 0.1% Tween 20 (TBST) and 5% low-fat milk powder at 25℃ for 1 hr, they were treated with anti-cleaved caspase-3 and -9 antibodies, as well as anti-β-actin antibody (Sigma) at 25℃ for 1 hr. The incubated membranes were washed five times with TBST for 5 min to eliminate nonspecific binding of the antibodies. After incubation with horseradish peroxidase-conjugated anti-IgG antibody (1:1,000) (Dako, Glostrup, Denmark) at 25℃ for 1 hr, immunoreactive spots were detected using ImmunoStar reagents (Wako) with the LAS-3000 instrument (FUJIFILM, Tokyo, Japan).
7
Protein Analysis of Mouse Skin Wound Healing
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Normal mouse skin tissue and mouse wound tissue 7 and 23 days post wounding were lysed in radioimmunoprecipitation (RIPA) buffer supplemented with a phosphatase inhibitor (PhosSTOP, Roche). The lysates were incubated for 20 min on ice and centrifuged at 20,000 × g for 15 min at 4°C. The supernatants were denatured at 95°C for 5 min in SDS sample buffer, consisting of 62.5 mmol/L Tris (pH 6.8), 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, and 0.001% bromophenol blue. Samples were separated by SDS–PAGE, and proteins were transferred onto a PDVF membrane (Bio-Rad, Munich, Germany). Membranes were incubated for 30 min at room temperature in blocking buffer consisting of 5% nonfat dry milk in phosphate-buffered saline (PBS) with 0.05% Tween-20, followed by an appropriate dilution of anti-POSTN Ab (Abcam) or anti-α-tubulin (Sigma) primary antibody overnight at 4°C. Immune complexes were detected with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, Piscataway, NJ), a chemiluminescence detection system (Perkin-Elmer, Waltham, MA), and a LAS-3000 instrument (Fujifilm, Tokyo, Japan)
8
Protein Extraction and Analysis Protocol
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Proteins were isolated using the NucleoSpin® RNA/Protein kit (Macherey-Nagel), according to the manufacturer’s protocol. The protein concentration was calculated using the BCA assay and bicinchoninic acid and copper (II) sulphate solutions (Sigma-Aldrich), and equal amounts of protein were separated by 8-12% SDS-PAGE and were electro-transferred to polyvinylidene fluoride (PVDF) membranes (ATTO, Tokyo, Japan). Immunoblots were probed with primary antibodies against the following proteins: FoxO3a (#2497, Cell Signaling, Danvers, MA, USA), HIF-1α (#14179, Cell Signaling), GATM (ab228937, Abcam, Cambridge, MA, USA), ALDH4A1 (ab185208, Abcam), ALDH1L1 (sc-100497, Santa Cruz), ALDH6A1 (sc-271582, Santa Cruz), COX IV (ab33985, Abcam), α-tubulin (#3873, Cell Signaling), and β-actin (A300-491A, Bethyl Laboratories, Montgomery, AL, USA). The primary antibodies were diluted 1:1,000 with 1% skim milk in PBS containing 1% Tween® 20 (Sigma-Aldrich) and incubated at 4°C overnight. Horseradish peroxidase-conjugated secondary antibodies in 2% skim milk were incubated with the blots for 1 h at room temperature. Immunoreactive proteins were detected using chemiluminescence reagent EzWestLumi Plus (ATTO) and visualized with a LAS-3000 instrument (Fujifilm, Tokyo, Japan).
9
In vivo Redox State Analysis of Redox Proteins
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In vivo redox states of redox proteins were analyzed using immunoblotting, as described previously (Mihara et al., 2018 (link)). Trx-m and FTR-C antibodies raised against the recombinant protein of Synechocystis sp. PCC 6803, polyclonal antibodies raised against recombinant Trx-m2, Trx-m3, Trx-x, Trx-y, TrxC, Alr2205, NTR, NTRC, and OpcA proteins of Anabaena 7120, GFP polyclonal antibody (Sigma, G1544), and GFP polyclonal antibody (Abcam, ab290) were used. The chemiluminescence of the horseradish peroxidase-conjugated secondary antibody was detected using a LAS 3000 instrument (Fuji Film, Tokyo, Japan).
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In vivo redox states of redox proteins were analyzed using immunoblotting, as described previously (Mihara et al., 2018 (link)). Trx-m and FTR-C antibodies raised against the recombinant protein of Synechocystis sp. PCC 6803, polyclonal antibodies raised against recombinant Trx-m2, Trx-m3, Trx-x, Trx-y, TrxC, Alr2205, NTR, NTRC, and OpcA proteins of Anabaena 7120, GFP polyclonal antibody (Sigma, G1544), and GFP polyclonal antibody (Abcam, ab290) were used. The chemiluminescence of the horseradish peroxidase-conjugated secondary antibody was detected using a LAS 3000 instrument (Fuji Film, Tokyo, Japan).
10
Western Blot Analysis of Protein Samples
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Cells were lysed in RIPA buffer containing a protease inhibitor cocktail (Roche, Madrid, Spain). After centrifugation, supernatants were collected and protein quantified using the DC protein assay (Biorad). Protein (30 µg/lane) was loaded on 10–12% SDS-poliacrylamide gels and transferred to PVDF membrane (iBlot® Get Transfer Stacks PVDF Regular, Invitrogen) using the iBlot® system (Invitrogen). Membranes were blocked and incubated O/N with primary antibodies (Table S1 ) diluted in 5% non-fat milk in PBS, 0.1% Tween 20 (Sigma). Next, PVDF membranes were incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Table S1 ). Finally, blots were developed with HRP SuperSignal® West Pico Trial (Thermo Scientific) and visualized in an LAS3000 instrument (Fujifilm, Madrid, Spain).
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