The denaturing gel of acrylamide 12% (SDS-PAGE) was used to separate the protein of interest. Samples were incubated at 95°C for 5 min with loading buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 0.01% bromophenol blue) and 100 mM DTT. 125 µg of total protein were loaded on gel next to the protein standard (Spectra Multicolor, Broad Range Protein Ladder, Thermo Scientific, MA, USA), and run at 100 V in run solution (25 mM Tris, 250 mM glycine, and 0.1% SDS). Proteins were transferred to Immobilon-P membrane (0.45 mm pore, Merck Millipore, Tullagreen, Carrigtwohill, Irland) with transfer solution (25 mM Tris, 192 mM glycine, 20% methanol) at 250 mA during 2 h.
Spectra multicolor
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Spectra Multicolor is a versatile lab equipment designed for spectroscopic analysis. It is capable of detecting and analyzing multiple wavelengths of light simultaneously. The core function of this product is to provide accurate and reliable data for various scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p membrane, by Merck Group (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Nupage lds sample buffer 4x, by Thermo Fisher Scientific (1 mentions)
Bio 500 professional vis gel scanner, by Serva Electrophoresis (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Luminata forte solution, by Merck Group (1 mentions)
4 protocols using spectra multicolor
1
SDS-PAGE Protein Separation and Western Blot
2
Casein Fractions Analysis by SDS-PAGE
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The extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli [39 (link)]. The assay was performed under denaturing conditions using a reducing agent to split the disulfide bonds. Briefly, 10 µL of the extracts was mixed with 10 µL of the sample buffer (NuPAGE LDS Sample Buffer 4X, Thermo Fisher). The mixture was heated to 95 °C for 5 min and then cooled to room temperature. 5 µL of each sample along with 5 µL of the broad range protein ladder (Thermo Scientific Spectra Multicolor, Size Range: 10 to 260 kDa) were loaded into the gels (NuPAGE™ 10%, Bis-Tris, 1.0 mm, Mini Protein Gel, 12-well; Thermo Scientific). The separation was performed at a constant current of 30 mA per gel for approximately 90 min, and the gels were then stained overnight using a Coomassie Brilliant Blue R-250 solution. After destaining for about 3 h with 10% acetic acid solution (the solution was changed regularly), the gels were scanned (Bio-500 Professional VIS Gel Scanner, SERVA Electrophoresis GmbH, Heidelberg, Germany) and densitometry analysis was performed to determine the relative concentrations of the casein fractions expressed in percent (ImageLab, Bio-Rad, Hemel Hempstead, UK).
3
Western Blot Analysis of Nav1.7 Protein
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Cells or tumor specimens were lysed using radio-immunoprecipitation assay (RIPA) buffer with the addition of protease and phosphatase inhibitor (Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations in each sample were quantified using a Pierce BCA Protein Assay Kit (Thermos Scientific, Waltham, MA, USA). Prior to performing gel electrophoresis, 1:1 of 2× Leammli Sample buffer (Bio-Rad, Hercules, CA, USA) was added to protein samples. The mixture was diluted with 5% 2-mercaptoethanol (ThermoFisher Scientific, Waltham, MA, USA). All protein samples were heated at 95 °C for 5 min and run on 4–15% Criterion TGX gradient gels (Bio-Rad). Gel transfer and immunoblotting detections were performed as previously described [57 (link)]. Primary antibodies for NaV1.7 (EMD Millipore Corp., Darmstadt, Germany) were used at 1:1000, and the reference protein GAPDH (Cell Signaling Technology, Danvers, CA, USA) and β-actin (Cell Signaling Technology) were used at 1:2000. Horseradish peroxidase-conjugated anti-rabbit/mouse with a dilution of 1:1000 (Cell Signaling Technology) was used as secondary antibodies. The molecular weight-marker broad-range protein ladder (10–260 kDa) (Spectra Multicolor, ThermoFisher Scientific) was used to confirm the size of the protein of our interest.
4
Rb44L1 Peptide Induces Apoptosis Signaling
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B16F10-Nex2 cells (106) were incubated with 0 and 130 μM of Rb44L1 peptide for different times (1, 3, 6, 8, and 24 h). After incubation, cells were washed in PBS and lysed with 300 μL of SDS sample buffer (62.5 mM Tris-HCl, pH 6.8 at 25°C, 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue). Proteins from whole cell extracts were analyzed by Western blotting as previously described (20 (link)). The following primary, highly specific monoclonal antibodies, were used: rabbit anti-Bcl-2, -Bcl-xl, -Bax, -caspase-9 and cleaved caspase-9, -caspase-3 and cleaved caspase-3, -Parp and cleaved Parp, and -GAPDH (for total protein loading control), with secondary anti-rabbit IgG conjugated with horseradish peroxidase (HRP). All antibodies were purchased from Cell Signaling Technology (Beverly, MA) except for anti-GAPDH, acquired from Sigma-Aldrich (St. Louis, MO). Immunoreaction was revealed using the Luminata™ Forte solution (Millipore, Billerica, MA) and images were acquired using Uvitec Cambridge (Cambridge, UK). The molecular mass of each protein was estimated based on a pre-stained protein standard (Spectra Multicolor, ThermoScientific, Waltham, MA). Full-length Western blotting membranes are displayed in Figure S1 .
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