After treatment, protein was isolated by resuspending cells in M-PER® Mammalian Protein Extraction Reagent (Thermo Scientific) and NE-PER lysate (Thermo Scientific) for immunoprecipitation with protease & phosphatase inhibitor cocktails (Thermo Scientific), incubated for 10 min at 4 °C, and centrifuged at 16,000 × g for 10 min to remove cell debris. Extracts were loaded onto 10% polyacrylamide gels and transferred to Hybond-ECL membranes (Amersham, Pollards Wood, UK). Membranes were blocked with 5% skim milk in phosphate-buffered saline containing 0.05% Tween 20 (PBST). The membranes were then incubated with specific primary antibodies diluted with PBST at 4 °C with gentle shaking overnight: p65, ATF3, topoisomerase II-α, IκBζ, α-tubulin and β-actin (Santa Cruz Biotechnology); ac-p65 (Abcam) and HDAC1 (Millipore). After several washings, the membranes were incubated with anti-mouse or anti-rabbit immunoglobulin G peroxidase-conjugated antibody (Thermo Scientific) diluted in PBST (1:1,000) for 2 h. After the membranes were washed several times with PBST, detection was carried out using Pierce ECL Western blotting substrate (Thermo Scientific) and exposure of the blots to a LAS-1000 system (Fuji, Tokyo, Japan). The intensities of protein bands were measured using the ImageJ software (http://rsb.info.nih.gov/ij/ )39 (link).
Las 1000 system
Manufactured by Fujifilm
Sourced in Japan
The LAS-1000 system is a laboratory equipment product from Fujifilm. It is designed for image capture and analysis. The system includes a high-resolution CCD camera, illumination unit, and software for acquiring and processing images.
Automatically generated - may contain errors
Lab products found in correlation
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Hybond ecl membrane, by Cytiva (1 mentions)
Protease phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Topoisomerase iiα, by Santa Cruz Biotechnology (1 mentions)
Ac p65, by Abcam (1 mentions)
Hdac1, by Merck Group (1 mentions)
α tubulin, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Phosphate buffered saline (pbs), by Fujifilm (1 mentions)
Anti pgk1, by Abcam (1 mentions)
Goat anti mouse poly hrp, by Thermo Fisher Scientific (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Anti myc 9e10, by Roche (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Anti pgk1, by Thermo Fisher Scientific (1 mentions)
A0545, by Merck Group (1 mentions)
15 protocols using las 1000 system
1
Western Blot Analysis of Protein Extraction
2
Immunoblot Analysis of Anti-Enolase Antibodies
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Nitrocellulose membranes (GE Healthcare, UK) were washed in PBS (Wako Pure Chemical Industries, Japan) containing 10 μg/ml recombinant α-enolase protein and 5 % (w/v) skim milk (BD Difco, USA) for 60 min at room temperature in non-reducing conditions. The membrane was washed three times with PBS containing 0.05 % Tween (Katayama Chemical Industries, Japan), and incubated with patient serum at a dilution of 1:200. After three washes in PBS containing 0.05 % Tween, the membrane was incubated with horseradish peroxidase–conjugated anti-human IgG antibodies at a dilution of 1:1000 (Sigma-Aldrich, USA), and peroxidase–conjugated mouse monoclonal antibodies to human IgG1, IgG2, IgG3, IgG4 at a dilution of 1:500 (Invitrogen, USA). After three washes, reaction product on the membrane was visualized using an enhanced chemiluminescence system (ChemiLumit Kit, GE Health Care, UK). Photographic images were obtained using an LAS-1000 system (FujiFilm Co., Japan). Image Reader Lite for LAS-1000 plus Ver. 1.3 (FujiFilm Co.) was used to capture the images, which were edited using Adobe Photoshop, when necessary.
3
Preparing Whole-Cell Extracts for Western Blotting
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Whole-cell extracts were prepared under denaturing conditions as described previously (Silve et al., 1991 (link)). Primary mouse monoclonal antibodies were diluted as follows: anti-myc 9E10 (Roche Applied Science), 1:5000; 3F10 anti-HA-HRP (Roche Applied Science), 1:5000; anti-myc-HRP 9E10 monoclonal antibody (Roche Applied Science), 1:5000; anti-Pgk1 (Abcam), 1:10,000. Where appropriate, goat anti-mouse–poly-HRP (Thermo Scientific; 1:5000) was used for immunodetection. Immunoreactive bands labeled were visualized by chemiluminescence detection (SuperSignal West Dura Extended-Duration Substrate; Thermo Fisher Scientific) of horseradish peroxidase using an LAS1000 system (Fuji Photo Film) or a ChemiDoc imaging system (Bio-Rad). Quantification was performed using ImageLab software (Bio-Rad).
4
Whole-Cell Extraction and Western Blotting
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Whole-cell extracts were prepared under denaturing conditions using NaOH and trichloroacetic acid as described previously (Silve et al., 1991 (link)). Cells were grown in SD medium, and when indicated (+), l -leucine (leu) was added at a concentration of 1.3 mM for 30 min to induce SPS-sensor signaling before extract preparation. Similarly, CHX (Sigma-Aldrich, St. Louis, MO) was added to the culture (CHX +) at a concentration of 100 μg/ml for the indicated time before extract preparation. Primary antibodies were diluted as follows: 3F10 anti–HA-horseradish peroxidase (Roche Applied Science, Basel, Switzerland), 1:2000; anti-Pgk1 (Molecular Probes), 1:1000; and anti-lexA (Abcam), 1:2000. Immunoreactive bands were visualized by chemiluminescence detection (SuperSignal West Dura Extended-Duration Substrate; Pierce, Rockford, IL) and quantified using the LAS1000 system (Fuji Photo Film, Tokyo, Japan).
5
TRPV1 Protein Expression Analysis
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Cells were lysed in RIPA buffer (#R0278, Sigma) supplemented with protease and phosphatase inhibitors (#P8340, #P5726, #P0044, Sigma). Protein concentration of lysates was determined by BCA assay (#23227, Thermo Scientific). 10 µg of proteins of cell lysates or TRPV1 control peptide (#ACC-030, alomone labs) were resolved in 10% acrylamide gels and transferred to PVDF membranes. Primary antibodies were incubated at 4 °C overnight (1:500, #TA336871, acris, or #ACC-030, alomone labs), followed by the secondary antibody (#A0545, Sigma, 1:5000, 1 h, room temperature (RT)). Signals were detected by chemiluminescence (#32209, Thermo scientific) using a LAS-1000 system (Fujifilm).
6
Cytokine Profiling in Bone Marrow-Derived Macrophages
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To investigate the cytokine profile in BMMs, they were cultured with 1 μg/ml of TgPLP for 24 hours in 6-well plates until 90% confluency. The supernatant of the cultured BMMs was collected and stored at −80°C until use. For the cytokine array, samples were analyzed using RayBio® Mouse Antibody Array-3 (RayBiotech Incorporation, Norcross, GA). The assay was performed according to the manufacturer's instructions. Briefly, the array membranes were incubated with blocking buffer for 12 hours at room temperature. After washing, the membranes were incubated with biotin-conjugated antibody for 12 hours at 4°C, and then incubated with HRP-conjugated streptavidin for 12 hours. The membranes were incubated with detection buffer and exposed by LAS 1000 System (Fuji, Japan). Finally, the scanned image was analyzed by the Multi-Gauge System (Fuji, Japan), and the signal intensity was calculated using ImageJ (NIH). After the normalization process (comparison between normal and positive control), data were represented as the relative expression of cytokines and chemokines [30 (link)].
7
Quantitative Yeast Protein Analysis
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Yeast whole cell lysates were generated by alkaline membrane lysis (15 min incubation on ice in 150 µL 1.85 M NaOH, 7.5% β-mercaptoethanol) and protein denaturation (≥10 min incubation on ice after addition of 150 µL 55% trichloroacetic acid and 1 mL water). Denatured proteins were pelleted at 4°C and 18000 g for 10 min. Pellets were washed with 150 µL 20 mM Tris (pH 7.4), 80% acetone, resuspended in HU-buffer (8 M urea, 5% SDS, 200 mM Tris–HCl, pH 6.8, 1 mM EDTA, 0.05% bromphenol blue, 4% β-mercaptoethanol), incubated for 30 min at 37°C, and separated by 7.5% SDS–PAGE followed by Western blotting using GFP-specifc antibodies (1∶20,000, rabbit serum, gift from Dirk Görlich, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany). Detection and quantification of protein bands from Western blots was done using the LAS-1000 system (Fujifilm) followed by image processing using MultiGauge (Fujifilm) and ImageJ software.
8
Comprehensive Western Blotting Protocol
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Western blotting was performed using standard procedures as described previously30 (link). A lysis buffer was supplemented with a complete mini protease inhibitor (Roche), ALLN (25 μg/ml), 0.5 mM NaF, and 10 μM Na3VO4 just prior to use. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Bio-Rad). All samples (20 μg of protein) were suspended in lysis buffer, fractionated using NuPAGE 4%–12% Bis-Tris (Invitrogen) gels, and transferred to a Protran nitrocellulose transfer membrane (Whatman). The membrane was blocked using 1× phosphate-buffered saline (PBS) containing 5% non-fat milk for 1 h and incubated with a primary antibody [anti-ABCA1 (1:1000), anti-ABCG1 (1:1000), anti-AMPKα (1:1000), anti-SREBP-1 (1:250), anti-TF2B (1:1000), anti-β-actin (1:3000), anti-CREB (1:1000), anti-PCK1 (1:1000), anti-SRC1 (1:200), anti-G6Pase (1:200) or anti-CPT1a (1:1000)] overnight at 4°C. After washing with PBS–0.05% Tween 20 (0.05% T-PBS), the membrane was incubated with a secondary antibody (anti-rabbit, anti-mouse and anti-goat IgG HRP-linked; 1:2000) for 1 h at 4°C. The membrane was then washed with 0.05% T-PBS and detected with an ECL Western Blotting Detection Reagent (GE Healthcare) using an LAS-1000 system (Fuji Film).
9
Immunoblotting for CYP2W1 detection
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Human and mice tissues were homogenized using the Bullet Blender (Next Advance, Averill Park, NY, USA) in a buffer containing 10 mM Tris–HCl pH 7.5, 0.5 mM EDTA, 0.25 M sucrose with addition of protease inhibitors cocktail (Roche Diagnostics, Mannheim, Germany). HCC2998 cells were lysed in RIPA buffer containing protease inhibitors cocktail for 30 min at 4°C. Denatured protein (40 μg) samples were run on 10% SDS-PAGE, transferred to nitrocellulose membranes and immunoblotted with two different rabbit anti-human CYP2W1 antibodies (H175, Santa Cruz, CA, USA, dilution 1:200 and the C-terminal in-house anti-CYP2W1-852 [4 (link)], dilution 1:1000), rabbit anti-mouse CYP2W1 antibodies (in-house anti-CYP2W1-3675, dilution 1:500), or with rabbit anti-ERp29 (in-house antibody reacting with both species [15 (link)], dilution 1:1000),javascript:void(0); followed by goat anti-rabbit conjugated horseradish peroxidase secondary antibodies (Dako, Glostrup, Denmark, dilution 1:2000). Filters were developed using SuperSignal West Femto Chemiluminescent Substrate (Pierce, Rockford, IL, USA) and signals detected by LAS-1000 system (Fujifilm, Japan).
10
Western Blot Analysis of Protein Extraction
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After treatment, proteins were isolated by resuspending the cells in M-PER mammalian protein extraction reagent (Thermo Scientific, Waltham, MA, USA) supplemented with the Halt protease inhibitor cocktail (Thermo Scientific). Extracts were loaded onto 10% polyacrylamide gels and transferred to Hybond enhanced chemiluminescence (ECL) membranes (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK). Membranes were blocked with 5% skim milk. The membranes were then incubated with specific primary antibodies, i.e., p-p53 (ser15) (1:1000) and β-actin (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and p53 (Millipore, MA, USA), which were diluted with PBS containing 0.05% Tween 20 (PBST), at 4°C with gentle shaking overnight. After several cycles of washing, the membranes were incubated for 2 h with ImmunoPure goat anti-mouse or anti-rabbit IgG peroxidase-conjugated antibody (Thermo Scientific) diluted in PBST (1:1,000). After washing several times with PBST, proteins were detected using the Pierce ECL western blotting substrate (Thermo Scientific). The blots were exposed to autoradiography films, which were analyzed with the LAS-1000 system (FUJIFILM).
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