The exosome pellet was dissolved in radioimmunoprecipitation assay lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) for 30 min at 4°C, and the protein concentration was determined using a Bradford protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) (17 (link),18 (link)). The proteins (20 µg/lane) were separated via 15% SDS-PAGE and then transferred to polyvinylidene difluoride membranes. Membranes were blocked in 5% non-fat dried milk for 1 h, followed by incubation for 1 h with anti-CD9 (1:1,000; cat. no. 13174, Cell Signaling Technology, Inc.) and anti-heat shock protein 90α (HSP90α; 1:1,000; cat. no. 8165, Cell Signaling Technology, Inc.) primary antibodies, and subsequent incubation for 1 h with the secondary antibodies (horseradish peroxidase-conjugated anti-rabbit immunoglobulin G; 1:1,000; cat. no. 7074, Cell Signaling Technology, Inc.). The bands were visualized using the SuperSignal chemiluminescence system (Thermo Fisher Scientific, Inc., Waltham, MA, USA). All steps were performed at room temperature.
Supersignal chemiluminescence system
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The SuperSignal chemiluminescence system is a laboratory equipment product designed for the detection and quantification of proteins and other biomolecules. It utilizes chemiluminescence technology to enable sensitive and accurate analysis.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Merck Group (2 mentions)
Chemilmager 4440, by Biozym (2 mentions)
Horseradish peroxidase conjugated anti rabbit immunoglobulin g, by Cell Signaling Technology (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Cell Signaling Technology (1 mentions)
Bradford protein assay kit, by Bio-Rad (1 mentions)
Ab8229, by Abcam (1 mentions)
Ab7613, by Abcam (1 mentions)
Ab18180, by Abcam (1 mentions)
Nb400 104, by Novus Biologicals (1 mentions)
Anti ha antibody, by Merck Group (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Sulfobiotics protein redox state monitoring kit plus, by Dojindo Laboratories (1 mentions)
Horseradish peroxidase linked secondary antibody, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Anti cd36, by Cayman Chemical (1 mentions)
Anti sr bi, by BD (1 mentions)
5 protocols using supersignal chemiluminescence system
1
Western Blot Analysis of Exosomes
2
Western Blot Protein Detection
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Proteins were isolated and equal amounts were separated by PAGE and transferred to nitrocellulose membranes (Sigma). After blocking, membranes were incubated with primary antibodies (anti-ABCA1, ab18180; anti-β-actin, ab8229; anti-apoA-I, ab7613 [all from Abcam, Cambridge, UK]; and anti-SRBI, NB400-104 [Novus]) at 4°C over night. Membranes were then incubated with the appropriate HRP-coupled secondary antibodies followed by detection using the Super Signal chemiluminescence system (Thermo Scientific, Rockford, IL) and a Chemilmager 4440 (Biozym, Oldendorf, Germany).
3
Western Blotting Protein Analysis Protocol
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Cellular lysates were resuspended, in a 4:1 ratio, in 4× NuPAGE LDS sample buffer (catalog number NP0007; Invitrogen) supplemented with 2-mercaptoethanol and heated at 70°C for 10 min in a heating block (Fisher Scientific). All samples were then loaded on Mini-Protean TGX precast gels (catalog numbers 4561034, 4561044, 4561084, and 4561094; Bio-Rad), and the denatured proteins were separated by electrophoresis. Protein was then wet transferred to polyvinylidene difluoride (PVDF) membranes (catalog number 1620177; Bio-Rad). Blots were blocked with Tris-buffered saline (TBS) supplemented with 0.1% Tween 20 with 5% milk for 1 h at room temperature, followed by 4°C overnight incubation with primary antibodies. The primary antibodies used included anti-FLAG antibody (catalog number F7425; Millipore), anti-HA antibody (catalog number H3663; Sigma-Aldrich), and anti-β-actin antibody (catalog number A2228; Sigma-Aldrich), all at 1:5,000 dilution, and anti-green fluorescent protein (GFP) antibody (catalog number GF28R; Invitrogen) at 1:1,000 dilution. The secondary antibodies used were anti-mouse antibody (catalog number NA931V; GE Healthcare) and anti-rabbit antibody (catalog number NA934V; GE Healthcare) at 1:5,000 dilution. The bands were detected using the SuperSignal chemiluminescence system (catalog number 34579; Thermo Scientific).
4
Monitoring Protein Redox States in Cells
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Alteration of protein redox state was assessed using the -SulfoBiotics- Protein Redox State Monitoring Kit Plus (Dojindo, Cat#SB12) for cellular proteins in accordance with the manufacturer's instructions (Dojindo Molecular Technologies Inc., Rockville, MD, USA) [34 ,35 ]. Briefly, to study the protein redox states in Pa03C cells, samples were prepared through protein precipitation using trichloroacetic acid (TCA) (Fisher, Cat#196057) and labeled with Protein-SHifter Plus upon non-reducing conditions in accordance with the manufacturer's instructions (Fig. 1 B and C). After cell extracts were subjected to SDS-PAGE, gels were exposed to UV light on a transilluminator to remove Protein-SHifter. Proteins in the gel were then electro-transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was blocked with 5% non-fat dry milk and incubated with Ref-1 antibody (Novus, Cat#13B8E5C2). Signals were obtained by using a horseradish peroxidase-linked secondary antibody (Bio-Rad, Cat#1706516) and the Super Signal Chemiluminescence System (Thermo, Cat#A38554) and bands were quantitated for Western blot band intensities.
5
Western Blot Analysis of Membrane Proteins
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Proteins were isolated and equal amounts were separated by SDS-PAGE and transferred to nitrocellulose membranes (Sigma). After blocking, membranes were incubated with primary antibodies (anti-SR-BI, BD Biosciences 610882; anti-β-actin, Abcam ab8229; anti-CD36, Cayman 100011) at 4°C over-night. Membranes were then incubated with the appropriate horseradish peroxidase-coupled secondary antibodies followed by detection using the Super Signal chemiluminescence system (Thermo Scientific) and a Chemilmager 4440 (Biozym, Oldendorf, Germany).
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