Cells were lysed with Nonidet P (NP)-40 buffer (1% NP-40, 0.15 M NaCl, 50 mM Tris; pH 8.0) containing protease inhibitors (Sigma-Aldrich; Merck KGaA). A bicinchoninic acid assay was used for protein quantitation (Pierce; Thermo Fisher Scientific, Inc.). Samples were denatured in SDS buffer and 50 µg protein was loaded per lane and separated by electrophoresis on SDS gels with 8–10% polyacrylamide. The proteins were transferred onto polyvinylidene difluoride membranes. These were then incubated with the following antibodies: Anti-PAPSS2 (cat. no. ab37611; 1:100) anti-COL2 (cat. no. ab185430; 2 µg/ml) and anti-COLX (cat. no. ab58632; 2 µg/ml), and anti-β-actin (cat. no. AC-40; 1:2,000; all Abcam, Cambridge, MA, USA) for 12 h at 4°C. Subsequently, membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary anti-mouse [m-IgGκ binding protein horseradish peroxidase (HRP) conjugated; cat. no. sc-516102] or anti-rabbit (mouse anti-rabbit HRP-IgG; cat. no. sc-2357) antibodies (each 1:5,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature for 2 h. Proteins were visualized using enhanced chemiluminescence (Advansta, Inc., Menlo Park, CA, USA) and the blots were imaged and quantified with the Fluor-S Multi-Imager system and Multi-Analyst software version 1.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Fluor s multi imager system
Manufactured by Bio-Rad
Sourced in United States, United Kingdom
The Fluor-S Multi-Imager system is a fluorescence imaging device designed for the detection and analysis of fluorescent molecules in various biological samples. The system utilizes a range of excitation sources and filters to capture high-quality images of fluorescently labeled samples, such as gels, blots, and microplates. The Fluor-S Multi-Imager system is capable of providing quantitative data and supports multiple fluorescent dyes and labels.
Automatically generated - may contain errors
3 protocols using fluor s multi imager system
1
Western Blot Analysis of Cartilage Proteins
2
Quantifying BBM Protein Expression
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Western blotting of the BBM was carried out as previously described (Marks et al. 2003 ) using rabbit polyclonal antibodies raised against SGLT1 (a kind gift from Professor G. Kellett, University of York, UK) and SGLT2 (a kind gift from Professor H Koepsell, University of Wurzburg, Germany). Mouse monoclonal antibody for β‐actin (ab6376, Abcam, Cambridge, UK) was used as a loading control. Blots were visualized with enhanced chemiluminescence on a Fluor‐S MultiImager system (Bio‐Rad, Hemel Hempstead, UK), and the abundance of each protein of interest was calculated relative to β‐actin and expressed as a percentage of the control average.
3
Proteome Profiling of Transgenic Tubers
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To examine the effect of AmA1 for the increase in total proteins were isolated from same mass (2 g) of WT and transgenic tubers and aliquots of 100 μl and 250 μl were subjected to 1-DE and 2-DE, respectively. Next, the proteome profiling of the WT and transgenic tubers was accomplished with 250 μg of protein using 2-DE analysis14 (link). The electrophoresed proteins were stained with Silver Stain Plus Kit (Bio-Rad). The gel images were scanned by the Fluor-S MultiImager system (Bio-Rad), and analyzed with PDQuest software version 7.2.0 (Bio-Rad). A match set representing a standard image of three replicates, from two biological replicates, was created for each sample. Each spot on the standard gel was quantified with several criteria for consistency in size and shape. The spots with a quality score <30 were eliminated from further analysis.
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