Single colonies of NPT110, carrying the lys2::ARM37 allele, were used to inoculate 1 ml of YP-Dextrose or YP-Galactose. Following overnight growth at 30°C, 10 μl of each culture was used to inoculate a fresh 1 ml of YP-Dextrose or YP-Galactose and cultures were grown to mid-log phase (approximately 6 hours). Cells were washed 1x in PBS and the pellets were resuspended in 50 μl of suspension buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.6, 1 mM EDTA, pH 8.0, 1 μg/ml aprotinin, 100 μg/ml PMSF). Next, 50 μl of 2x loading buffer (100 mM Tris-HCl, pH 6.8, 200 mM DTT, 4% SDS, 0.2% bromophenol blue, 20% glycerol) was added and samples were incubated for 10 minutes at 100°C. The insoluble material was pelleted and 20 μl of the supernatant was resolved on a 12% SDS-PAGE gel at 100V using the BioRad mini PROTEAN system. Protein was then transferred to PVDF membrane using the BioRad mini Trans-Blot cell running at 150 mA for 1 hour. The membrane was blocked with a solution of 5% dry milk dissolved in TBST (20 mM Tris-HCl, pH 7.5, 150mM NaCl, 0.05% Tween-20) and blotted with anti-myc antibody. Signal was produced by using the ECL Prime Western Blotting Detection Kit (GE Healthcare) and detected using the BioRad Fluor-S MultiImager.
Fluor s multiimager
Manufactured by Bio-Rad
Sourced in United States, United Kingdom
The Fluor-S MultiImager is a gel documentation and analysis system designed for imaging a variety of fluorescent and chemiluminescent samples, including DNA, protein, and cell-based assays. The system utilizes a CCD camera and various excitation and emission filter sets to capture high-quality images of the samples.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one software, by Bio-Rad (11 mentions)
Luminol reagent, by Thermo Fisher Scientific (5 mentions)
Multi analyst software, by Bio-Rad (3 mentions)
0.2 μm nitrocellulose membrane, by Bio-Rad (2 mentions)
Actin a2066, by Merck Group (2 mentions)
Immobilon p membrane, by Merck Group (2 mentions)
Pan py, by Cell Signaling Technology (2 mentions)
Cbl py674, by Abcam (2 mentions)
Itk ptyr512, by Thermo Fisher Scientific (2 mentions)
Lat py255, by Thermo Fisher Scientific (2 mentions)
Amersham ecl prime reagent, by GE Healthcare (2 mentions)
Supersignal west femto chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Anti actin antibody, by Merck Group (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Ecl prime western blotting detection kit, by GE Healthcare (1 mentions)
Mini trans blot cell, by Bio-Rad (1 mentions)
Mini protean system, by Bio-Rad (1 mentions)
Goat anti mouse igg hrp 7076s, by Cell Signaling Technology (1 mentions)
48 protocols using fluor s multiimager
1
Galactose-Induced Protein Expression Analysis
2
Western Blot Quantification of Ac45 Protein
3
Western Blot Analysis of Mandibular Root Proteins
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Mandibular root samples from 4-day and 14-day WT and Atp6i−/− mice were dissected and rinsed in chilled PBS, and immediately frozen at −80 °C until used for protein extraction. For preparing the samples for western blot analysis, we harvested the root samples cutting from the alveolar bone-root region of the WT mice mandible, which included root, dental pulp and alveolar bone surrounding the root and exclude crown. As there is no tooth root for Atp6i−/− mice, we harvested the root samples from the same area as from the WT mice in order to compare the protein expression level of the same alveolar bone-root area. Protein extraction from root samples or odontoblasts and western blotting were performed as previously described50 ,53 (link) and a Fluor-S Multi-Imager with Multi-Analyst software (Bio-Rad) was used for visualization and quantification. The rabbit anti-Atp6i antibody was previously generated by our lab7 (link) and was used at a 1:1 000 dilution. phospho-Smad2/3 protein and Smad2/3 protein levels were analyzed with the following primary antibodies: rabbit-anti-phospho-Smad2/3 (1:1 000; Cell signaling) and rabbit-anti-Smad2/3 (1:1 000; Cell signaling). Horseradish peroxidase-linked anti-rabbit IgG (7074 S, Cell signaling) was used to visualize the reaction.
4
Cell Lysis and Protein Immunoblotting
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Cells were lysed on ice for 30 min in RIPA buffer (CST, UK) containing protease and phosphatase inhibitor cocktails (Sigma, UK). Lysates were clarified by centrifugation (13,000 rpm, 15 min, 4°C), diluted with Laemmli buffer, and denatured at 95°C for 5 min. SDS-PAGE and immunoblotting were performed according to standard protocols [49 ]. Most primary antibodies used were from CST, BAG-1 (3.10 G3E) was from Santa Cruz Biotechnology, and β-actin-HRP was from Sigma (UK). HRP-conjugated secondary antibodies were from Dako (Denmark). Proteins were detected by SuperSignal West Pico or Fempto chemiluminescent substrate (Thermo Scientific, UK) using a BioRad Fluor-S Multiimager with Quantity One 1-D v4.6.6 image acquisition and analysis software.
5
Proteomic Analysis of Differential Protein Expression
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Gel images were captured using the Fluor-S Multi-Imager (BIO-RAD) and analyzed by PDQuest Advanced 8.0.1 2D Gel Analysis Software (version 7.0, BioRad, Hercules, CA, USA Bio-Rad). 2D gel profiles were screened for DAPs identification. The images were cropped to the same size and shape, and spots were detected and matched automatically to a master gel selected by the software. The analysis comprised spot detection, gel matching and statistical analysis. Spot detection and matching were edited manually. The separate analysis of each sample included alignment of each treated gels to its reference image (control gels). Quantitative analyses were carried out after normalizing spot volumes in all gels to compensate for abundance-related variations. Selection of DAP spots was based on fold change abundance > 2.0, with a consistent change in the three technical replicas of the two biological extracts.
6
Protein Extraction and Immunoblotting Protocol
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Protein extraction and immunoblotting were performed as previously described [47 (link)]. Primary antibodies were as follows: anti-Notch1 (Clone A6 Novus Biologicals, Cambridge, UK), anti-Thbs1(sc-73158, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-E-Cadherin (Clone NCH-38, Dako, Denmark) anti-mTor (2993, Cell Signaling Technology, Beverly, MA), anti-Icam5 (Abcam, Cambridge, UK), anti Pai3 (sc-99153, Santa Cruz Biotechnology), anti-Vimentin (Clone V9, Dako), anti Ck19 (Clone RCK108, Dako), anti-Ck18 (Clone DC10, Dako), anti-Mmp-9 (Clone 6-6B, Calbiochem, San Diego, USA), anti-Snail (sc-28199, Santa Cruz Biotechnology), anti-Ck8 (sc-52324, Santa Cruz Biotechnology), anti-Alpha-Sma (Clone 1A4, Sigma), anti-E-Cadherin (Clone 4A2, Cell Signaling) and anti-β-Actin monoclonal antibody (Clone AC-40). Immunoreactivities were revealed with the EnVision dextran polymer visualization system (Dako). Membranes were washed and autoradiographies were obtained using a chemiluminescence reaction (ECL reagents, Amersham). Digital images of autoradiographies were acquired with a scanner (Fluor-S MultiImager, Bio-Rad) and signals were acquired in the linear range of the scanner and quantified using specific densitometric software (QUANTITY-ONE, Bio-Rad) in absorbance units.
7
Western Blot Analysis of Protein Samples
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Briefly, for WB analysis, cultured cells were lysed in ice-cold lysis buffer. Proteins were separated using 10% gel, transferred to 0.2-μm nitrocellulose membranes (Bio-Rad, Hercules, CA, USA) and visualized using standard immunoblotting protocol. All antibodies were diluted in Tris-buffered saline (TBS) containing 5% (w/v) nonfat dry milk. Blots were developed using the Luminol reagent (Thermo Fisher Scientific) and densitometric analysis was performed as described previously, using a Fluor-S MultiImager with Quantity One software (Bio-Rad).40 (link), 41 (link)
8
Western Blot Analysis of Extracellular Matrix Proteins
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Proteins were extracted from cultured cell lysates with phosphatase and protease inhibitor cocktails. Protein (20 µg) was separated from each sample by 6% or 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membranes were blocked using 5% non-fatty milk in PBS containing 0.1% Tween-20 for 1 h at RT, and were then incubated with primary antibodies overnight at 4 °C. Subsequently, the membranes were washed with PBS containing 0.1% Tween-20 three times and then incubated with secondary antibodies conjugated with horseradish peroxidase for 90 min at RT. Protein bands were detected using a Fluor-S MultiImager (Bio-Rad, Hercules, CA). The band density was measured using NHI Image software (version 1.53; NIH, Bethesda, MD); β-actin served as a loading control. The antibodies used in the analysis were anti-Col-I (ab90395), anti-α-SMA (ab7817) and anti-OPN (ab8448) (Abcam, Cambridge, UK).
9
Protein Separation and Immunoblotting
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Extracted proteins were separated by SDS–PAGE and immunoblotting was carried out as described before [2] (link). Briefly, primary antibodies were incubated overnight at 4°C. Then the membranes were rinsed with TBST (0.1% Tween-20 in 0.01 M TBS). The blots were incubated with appropriate secondary antibody at room temperature for additional 1 h. Protein bands were visualized using an enhanced chemiluminescence system (Supersignal West Pico Trial Kit, Pierce, Rockford), and the density of each band was quantified with a Fluor-S MultiImager (Bio-Rad). The experiment was repeated in triplicate.
10
Western Blotting Quantification and Antibody Detection
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