HFFs were cultivated to confluency in six-well plates and 250 parasites were inoculated and cultured at 37°C for 11 days without disturbance. After washing with 1 mM D-PBS, the culture was fixed with methanol (Sigma-Aldrich) at 4°C for 20 min. The fixed culture was treated with 0.25% CBB R-250 (Wako) for 3 h. The images of plaques were procced on ImageJ software (http://imagej.nih.gov/ij/ ) by calculating the area size of all plaques. The plaque area was defined manually and marked with lines to major the area size by using Tools in Polygon Section.
Cbb r 250
Manufactured by Fujifilm
Sourced in Japan
The CBB R-250 is a laboratory equipment product from Fujifilm. It is designed for performing electrophoresis techniques. The core function of this product is to facilitate the separation and analysis of macromolecules, such as proteins and nucleic acids, in a controlled and standardized environment.
Automatically generated - may contain errors
6 protocols using cbb r 250
1
Plaque Quantification for Parasite Infection
2
SDS-PAGE Analysis of NNV Particles
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Before performing SDS − PAGE, NNV particles were suspended in SDS-denature buffer (2% [w/v] SDS, 0.05% [v/v] ß-mercaptoethanol in 25 mM Tris-HCl [pH 6.8]) and boiled for 3 min. After cooling, tracking dye and glycerol mixture (0.5% [w/v] bromophenol blue and 70% [v/v] glycerol in 25 mM Tris-HCl [pH 6.8]) were added to each denatured sample. SDS − PAGE was performed under reducing conditions according to Laemmli’s method23 (link) using 10% polyacrylamide gel. Gel proteins were stained with CBB (R-250, Wako). Density profiles for digital images of NNV CP, BSA and molecular weight markers in SDS–polyacrylamide gel were analyzed using ImageJ software version 1.53 (National Institutes of Health, Bethesda, Maryland, USA)24 (link).
3
Protein Separation and Visualization
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4
Quantitative Western Blot Analysis
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Seven micrograms of protein extract from each sample were electrophoresed in duplicate on 12.5% SDS-polyacrylamide gels53 (link). One gel was stained with Coomassie Brilliant Blue stain (CBB R-250, Wako, Japan) and the second was used for western blotting; the bands were transferred to a polyvinylidene difluoride membrane. For primary antibody incubation, 1:10000 diluted mouse anti-MDP1 antibody (7C monoclonal antibody) was used and anti-heat shock protein Hsp65 (GroEL2) antibody was used as an internal control23 (link). Reactions were visualized using ECL Prime western blotting detection reagent (GE Healthcare Lifesciences, Japan). The fractionation patterns were compared using the public domain software ImageJ (a Java image processing program developed by the National Institutes of Health).
5
SDS-PAGE Protein Separation and Visualization
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Protein solutions containing 5 to 20 µg protein were mixed with sample buffer containing 2% sodium dodecyl sulfate (SDS; Bio-Rad Laboratories), 50 mM Tris-HCl (pH 6.8), 6% 2-mercaptoethanol (Wako Pure Chemical, Osaka, Japan), 10% glycerin (Wako Pure Chemical), and 0.04% CBB-R250 (Wako Pure Chemical), followed by boiling at 100°C for 3 min. Samples were electrophoresed on a 10% or 15% polyacrylamide gel in the presence of 0.1% SDS and 0.192 M glycine (Bio-Rad Laboratories) using a 0.025 M Tris-glycine buffer (pH 8.3). The conditions were a fixed voltage of 100 V for 5 min, followed by a current of 30 mA for 40 min, and dyeing was performed using Coomassie Brilliant Blue (CBB)-R250 solution.
6
Photocatalytic Protein Profiling of AD Mouse Brain
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The cortex and hippocampus from a 7-month-old AppNL-G-F/NL-G-F mouse brain were combined and homogenized using 10× volume of tris buffer [50 mM tris, 150 mM NaCl (pH 7.6), cOmplete EDTA+ (Millipore Sigma, St. Louis, USA)]. Catalyst 7 or riboflavin (50 μM) was added to the lysate, and the mixture was irradiated with 595- or 500-nm light for 2 hours at room temperature. After the reaction, the lysate was centrifuged (260,000g, 20 min, 4°C) to obtain a soluble supernatant fraction, and the fraction was analyzed by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) with coomassie brilliant blue (CBB) staining [0.1% CBB R-250 (FUJIFILM Wako Pure Chemical Corporation)/50% MeOH and 10% AcOH].
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