Plasmids were introduced into protoplasts by polyethylene glycol-mediated transformation47 . Protein extracts were prepared at 24 h after transformation or at the indicated time points48 . The protoplasts were treated with tunicamycin (10 μg/mL; Sigma-Aldrich, St. Louis, MO) immediately after transformation. Cycloheximide (50 μg/mL; Sigma-Aldrich, St. Louis, MO) treatment was applied at 12 h after transformation. Protein extracts were analyzed by western blotting with anti-HA (Roche Diagnostics, Indianapolis, IN), anti-actin (MP Biomedicals, Solon, OH), anti-GFP (Bio-Application, Pohang, Korea), or anti-BiP antibodies47 –49 . The protein blots were developed with an ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ), and images were obtained using an LAS4000 image analyzer (Fujifilm, Tokyo, Japan). To quantify expression levels, intensity of protein bands in western blot images was measured using Multi-Gauge program equipped to LAS4000 image analyzer (FUJIFILM, Tokyo, Japan). Band intensities were added in cases of glycosylated proteins except the bottom band that was considered as the non-glycosylated form.
Las 4000 image analyzer
Manufactured by Fujifilm
Sourced in Japan
The LAS-4000 is a compact image analyzer designed for life science applications. It features a high-resolution CCD camera, advanced image processing capabilities, and a user-friendly software interface. The LAS-4000 allows for the capture, analysis, and quantification of a wide range of biological samples, including gels, blots, and microplates.
Automatically generated - may contain errors
Lab products found in correlation
Las 4000, by Cytiva (15 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (4 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (3 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (3 mentions)
Peroxidase conjugated secondary antibody, by Nichirei Biosciences (3 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (3 mentions)
Lumi lightplus western blotting kit, by Roche (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (2 mentions)
West zol plus western blotting detection system, by iNtRON Biotechnology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene difluoride membrane, by GE Healthcare (2 mentions)
P iκbα, by Cell Signaling Technology (2 mentions)
Imagequant las 4000, by GE Healthcare (2 mentions)
Iblot transfer stack pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Anti gfp antibody, by Roche (2 mentions)
Chemiluminescent substrate, by PerkinElmer (2 mentions)
Anti actin, by Merck Group (2 mentions)
Horseradish peroxidase, by Merck Group (2 mentions)
Anti transferrin receptor, by Zymo Research (2 mentions)
46 protocols using las 4000 image analyzer
1
Protein Expression Analysis in Protoplasts
2
Western Blot Analysis of Cellular Signaling
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Western blot analysis was performed using the method described previously21 (link). Whole-cell lysates and plasma membrane fractions were subjected to SDS-PAGE, and the proteins that had migrated were electrically transferred to a microporous polyvinylidene fluoride membrane (Millipore Corporation, MA, USA) for western blotting21 (link). The membrane was incubated at 4 °C for 18 h with antibodies against the following proteins: p-Akt, Akt, p-AMPK, AMPK, p-ACC, ACC (all at 1:1000), and β-actin (1:2000)21 (link). After incubation, anti-mouse (1:2000 for β-actin) or anti-rabbit IgG horseradish peroxidase conjugate (1:2000 for p-Akt, Akt, p-AMPK, AMPK, p-ACC, and ACC) was added, and the membrane was incubated for 30 min at RT. The antigenic proteins on the membrane were visualized via chemiluminescence using a Lumi-LightPLUS Western Blotting Kit (Roche Diagnostics Co., Basel, Switzerland), and the images were evaluated using an Image Analyzer LAS-4000 (Fujifilm Co. Tokyo, Japan)21 (link).
3
Western Blot Analysis of Signaling Proteins
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Whole-cell lysates and plasma membrane fractions were subjected to SDS-PAGE, and the proteins that had migrated were electrically transferred to a polyvinylidene fluoride (PVDF) microporous membrane (Millipore Corporation, MA, USA) for Western blotting. The membrane was incubated at 4°C for 18 h with antibodies against the following proteins: p-Akt, Akt, p-AMPKα, AMPKα, p-ACC, ACC, p-LKB1, LKB1, p-PDK1, PDK1, GLUT4 (1∶1000), E-cadherin (1∶200), and β-actin (1∶2000). After the incubation, anti-mouse IgG horseradish peroxidase-conjugate (HRP, 1∶2000, for β-actin) or anti-rabbit IgG HRP (1∶2000, for p-Akt, Akt, p-AMPKα, AMPKα, p-ACC, ACC, p-LKB1, LKB1, p-PDK1, PDK1, and GLUT4) was added; and the membrane was incubated for 30 min at room temperature. The antigenic proteins on the membrane were visualized by chemiluminescence by use of a Lumi-LightPLUS Western blotting kit (Roche Diagnostics Co., Basel, Switzerland), and the images were evaluated by using an Image Analyzer LAS-4000 (Fujifilm Co. Tokyo, Japan).
4
Western Blot Analysis of Cytoplasmic Proteins
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Cytoplasmic lysates were obtained with the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Waltham, MA, USA) and 35 µg of total proteins were resolved by SDS-PAGE (12%, reducing conditions), electrotransferred onto PVDF membranes, blocked for 1 h with 5% non-fat milk in 0.1% PBS/Tween 20 and incubated overnight at 4 °C with indicated antibodies at 0.1 µg mL -1 in 10 mL of blocking solution. Membranes were washed and incubated for 1 h with horseradish peroxide-conjugated secondary antibody (1/50 dilution) for 1 h at room temperature. Membranes were developed by chemiluminiscence with the ECL Prime Western Blotting Detection Reagent (Amersham, Chicago, IL, USA). Light emission was detected with a digital imaging system (Fujifilm Image Analyzer LAS-4000, Tokyo, Japan) and analyzed with the Multi Gauge software. Rehybridization of membranes with the anti-β-actin mAb was used as loading controls.
5
Immunoblot Analysis of MAPK Signaling
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Immunoblot analysis was performed as previously described [18 (link)]. Total cell lysates were prepared and subjected to immunoblot analysis using the following antibodies: anti-ERK1/2 (1:1000), anti-phospho-ERK1/2 (1:1000), anti-MAPK/ERK kinase 1/2 (MEK1/2) (1:1000), anti-phospho-MEK1/2 (1:1000), anti-phospho-Raf-1 (Ser-338) (1:1000), anti-EGFR (1:1000), anti-phospho-EGFR (Tyr-1068) (1:1000) (Cell Signaling Technology Inc., Beverly, MA, USA), anti-Raf-1 (1:1000) (BD Transduction Laboratories, BD Biosciences Inc., San Jose, CA, USA), or anti-involucrin (1:1000) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The luminescent signals were analyzed using an LAS-4000 image analyzer (Fuji Film Co., Tokyo, Japan). Immunoblot band intensities were quantified using ImageJ software (NIH, Bethesda, MD, USA).
6
HBV DNA Detection Protocol
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The isolated core‐particle‐associated HBV DNA was separated on a 1.0% agarose gel and transferred to a nylon membrane (positively charged; Roche Diagnostics, Penzberg, Germany). The transferred membrane was immobilized by an ultraviolet crosslinker and hybridized by an alkaline‐ phosphatase‐labeled probe of a full‐length HBV DNA fragment (accession number AB246344, https://www.ncbi.nlm.nih.gov/nuccore/AB246344 ) generated with the Gene Images AlkPhos Direct labeling system (DIG‐High Prime DNA Labeling and Detection Starter Kit II; Roche Diagnostics). Chemiluminescent detection was performed with CDP‐Star (DIG‐High Prime DNA Labeling and Detection Starter Kit II) and analyzed using an LAS4000 image analyzer (Fuji Photo Film, Tokyo, Japan).
7
Analyzing Tau Phosphorylation in DYRK1A Mice
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DYRK1A transgenic mice with three copies of the human DYRK1A gene were maintained as described previously (Ahn et al., 2006 (link)). Experiments were performed in accordance with the guidelines under the approval of the Institutional Review Committee for Animal Care and Use, KRIBB, Daejeon, Korea. The 8- to 10-week-old C57BL/6 mice were administered with DMSO or CX-4945 (75 mg/kg of body weight) orally in PBS solution, and the hippocampus was dissected after 30 min. The hippocampal lysates were prepared by using a Digital Sonifier Cell Disruptor instrument (BRANSON) in CelLytic MT buffer (catalog # C3228, Sigma-Aldrich) containing protease inhibitor cocktail set III (catalog # 535140-1ML, Calbiochem). The protein concentration was determined using the Bradford protein assay (Bio-Rad). Proteins were separated by SDS-PAGE and analyzed by immunoblotting with antibodies against phosphorylated Tau and Tau using an LAS4000 image analyzer (Fujifilm, Tokyo, Japan).
8
Western Blotting of Key Proteins
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Western blotting was performed as described elsewhere10 (link),11 (link) with the following antibodies: anti-AKT3 (Sigma-Aldrich Co., St Louis, MO, USA), anti-PSAT1 (Sigma), and anti-GAPDH (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Chemiluminescence signals were visualized with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) in a LAS-4000 image analyzer (Fuji Photo Film, Japan). The expression levels of these proteins were evaluated by Quantity One software.
9
Western Blot Protocol Characterization
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Cells were lysed with lysis buffer [52.5 mM Tris-HCl (pH6.8), 2.15% sodium dodecyl sulfate, 5% β-mercaptoethanol, 7% glycerol, and 0.0025% bromophenol blue]. Cell lysates were subjected to SDS-PAGE followed by conventional wet transfer. Membranes were incubated with antibodies and exposed to secondary horseradish peroxidase-conjugated antibodies (Cell Signaling Technology, Danvers, MA, USA, and Invitrogen, Waltham, MA, USA). Images were detected using an LAS-4000 image analyzer (Fujifilm, Tokyo, Japan). Antibodies used in this study are listed in Supplementary Table S1 .
10
Western Blot Analysis of EMT Markers
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Proteins from tissues and whole-cell lysates were prepared with radio immunoprecipitation (RIPA) buffer containing phenylmethane-sulfonylfluoride (PMSF; Sigma-Aldrich, St Louis, MO, USA) and 1 × complete protease inhibitor cocktail tablets (Roche, Indianapolis, IN, USA). Total proteins were separated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was blocked with blocking buffer (5% skimmed milk in Tris-buffered saline, 0.1% Tween-20) and incubated with the following antibodies: anti-TCIRG1 (ab139812), anti-Snail (ab78105) (both from Abcam, Cambridge, MA, USA), anti-GAPDH (sc-32233), anti-fibronectin (sc-29011) (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-E-cadherin (#610181), anti-N-cadherin (#610920) (both from BD Transduction, San Jose, CA, USA), anti-Vimentin (#5741) and anti-Slug (#9585) (both from Cell Signaling Technology, Danvers, MA, USA). Chemiluminescence signals were detected with Immobilon horseradish peroxidase substrate (Millipore, Billerica, MA, USA) and visualized using an LAS-4000 image analyzer (Fuji Photo Film, Tokyo, Japan).
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