Sample protein concentrations were measured using the PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), and an equal amount of protein (10 µg) of each sample was separated by SDS-PAGE by using NuPAGE Sample Reducing Agent (Thermo Fisher Scientific, Waltham, MA, USA) on precast NuPAGE 4–12% Bis-Tris Protein Gels (Thermo Fisher Scientific, Waltham, MA, USA). Thereafter the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and probed using anti-Calnexin (ab22595, Abcam, Cambridge, UK), anti-Alix (ab117600, Abcam, Cambridge, UK) or Syntenin-1 (TA504796, OriGene, Rockville, MD, USA) antibodies as per manufacturer’s instructions. The secondary antibodies used were goat anti-mouse IRDye 800 CW (926-32210, LI-COR Biosciences, Lincoln, NE, USA) or donkey anti-rabbit IRDye 800 CW (926-32213, LI-COR Biosciences, Lincoln, NE, USA). Precision Plus Protein Dual Color Standards ladder was utilized to determine the size of protein bands (Bio-Rad Laboratories, Hercules, CA, USA). The final blots were imaged using the LI-COR Odyssey CLx infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA).
Precision plus protein dual color standards ladder
Manufactured by Bio-Rad
Sourced in United States, United Kingdom
Precision Plus Protein Dual Color Standards ladder is a pre-stained molecular weight marker used for protein electrophoresis. It provides a visual reference for tracking the migration of proteins during SDS-PAGE.
Automatically generated - may contain errors
Lab products found in correlation
Nupage sample reducing agent, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab117600, by Abcam (1 mentions)
Irdye 800cw donkey anti rabbit, by LI COR (1 mentions)
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Ab22595, by Abcam (1 mentions)
Irdye 800cw goat anti mouse, by LI COR (1 mentions)
Odyssey clx infrared imaging system, by LI COR (1 mentions)
Claudin 3, by Abcam (1 mentions)
Trans blot turbo pvdf transfer kit, by Bio-Rad (1 mentions)
Alexafluor igg secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Occludin, by Novus Biologicals (1 mentions)
β actin, by Merck Group (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Odyssey fc imaging system, by LI COR (1 mentions)
Mini protean tgx precast protein gel, by Bio-Rad (1 mentions)
5 protocols using precision plus protein dual color standards ladder
1
Quantitative Protein Expression Analysis
2
Protein Expression Analysis of Colonoid Samples
3
Quantitative Protein Analysis by SDS-PAGE
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EV sample total protein concentrations were determined by BCA assay (Thermo Fisher Scientific, Loughborough, UK), and 10 µg of protein separated by SDS-PAGE using NuPAGE Sample Reducing Agent and precast NuPAGE 4–12% Bis-Tris Protein Gels (both Thermo Fisher Scientific). Precision Plus Protein Dual Color Standards ladder was utilized to determine the size of protein bands (Bio-Rad Laboratories, Perth, UK). Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and probed with antibodies as appropriate (details of primary and secondary antibodies are provided in Table S2). Chemiluminescence was measured using the Odyssey Fc Imaging System (LI-COR Biosciences, Cambridge, USA).
4
Protein Quantification and Western Blot Analysis
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The protein concentrations of the samples were determined using a Pierce BCA Protein Assay Kit from Thermo Fisher Scientific. Standard SDS-PAGE was performed using both the sarkosyl-soluble and sarkosyl-insoluble fractions derived from cell and AD brain extractions. The samples were loadd on a 4–20% Mini-PROTEAN® TGX™ Precast Protein Gel from Bio-Rad. Precision Plus Protein Dual Color Standards ladder from Bio-Rad was used as a reference for molecular weights. For the western blot staining, the MC1 antibody hybridoma conditioned media was employed, which was kindly provided by the lab of Peter Davies. The immunoblots were imaged using a Licor Gel Imaging Station and subsequently analyzed with Fiji imaging software.
5
Quantitative Western Blot Analysis
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Biomass concentrations in frozen cell pellets were equalized by adding appropriate volumes of Lämmli buffer (0.0625 M Tris–HCl (VWR) pH = 6.8, 2% SDS (Merck), 10% glycerol (Biosolve Chimie SARL,), 0.1 M dithiothreitol (Sigma-Aldrich) and 0.001% bromophenol blue (Fisher Scientific)). Samples were kept on ice, heated for 5 min at 99 °C and centrifuged at 20,000 rcf for 5 min before loading. SDS-PAGES were hand-casted (4% stacking gel, 12% separating gel). Proteins were separated by electrophoresis and transferred to a 0.2 µm nitrocellulose membrane (Bio-Rad). sfTq2ox was detected with primary antibody αGFP (1:2000, Fisher Scientific), FtsZ with a polyclonal antibody generated in rabbit against E. coli FtsZ35 (link). Both rabbit-antibodies were subsequently detected with the secondary Peroxidase AffiniPure Donkey Anti-Rabbit IgG (H + L) (Jackson ImmunoResearch). mNG was detected with anti-mNG (Chromotek) and secondary goat anti-mouse IgG (H + L) HRP (Invitrogen). The conjugated horse radish peroxidase (HRP) of the secondary antibody was subsequently visualised with the Pierce™ ECL Western blotting substrate (Thermo Scientific) in a Licor Odyssey machine (700 nm channel for the Precision Plus Protein Dual Color Standards ladder (Bio-Rad), chemiluminescence channel for HRP activity).
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