Immunoblot was performed according to standard procedures [14 (link),17 (link)]. Briefly, proteins were extracted from cells with PhosphoSafe Extraction Reagent (Merck KGaA, Darmstadt, Germany) and quantified using bicinchoninic acid assay (Thermo Scientific, Hennigsdorf, Germany). Proteins were electrophoresed on 8–10% SDS-PAGE gel and transferred to a PVDF membrane (Merck Millipore, Darmstadt, Germany). Membranes were incubated with primary antibodies overnight at 4 °C and with secondary antibodies for 2 h at room temperature. Binding was detected using Radiance Plus Chemiluminescent substrate (Azure Biosystems, Dublin, CA, USA) and visualized using CHEMOSTAR ECL Imager (INTAS Science, Göttingen, Germany). All antibodies used are listed in Supplementary Table S4 .
Radiance plus chemiluminescent substrate
Manufactured by Azure Biosystems
Sourced in United States
Radiance Plus is a chemiluminescent substrate produced by Azure Biosystems. It is designed for use in Western blot and other protein detection applications that utilize chemiluminescence detection methods.
Automatically generated - may contain errors
Lab products found in correlation
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Phosphosafe extraction reagent, by Merck Group (1 mentions)
Ibright imaging system, by Thermo Fisher Scientific (1 mentions)
β actin, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Phosstop, by Roche (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Skimmed milk, by Nacalai Tesque (1 mentions)
Pierce bca assay kit, by Thermo Fisher Scientific (1 mentions)
Mini trans blot cell, by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Goat anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Azure c600 gel imaging system, by Azure Biosystems (1 mentions)
Ab109508, by Abcam (1 mentions)
Ab8245, by Thermo Fisher Scientific (1 mentions)
Ab109497, by Cell Signaling Technology (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
α tubulin, by Abcam (1 mentions)
5 protocols using radiance plus chemiluminescent substrate
1
Immunoblot Analysis of Cellular Proteins
2
Protein Isolation and Western Blot Analysis
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Protein was isolated in EBC (50 mM Tris‐HCl, 120 mM NaCl, 0.5% NP‐40, pH 8.0) lysis buffer containing Complete Protease Inhibitor Cocktail (Roche) and PhosSTOP (Roche). Protein concentrations were quantified using Coomassie blue staining and a bovine serum albumin (BSA) standard. Protein (10−20 µg) containing dithiothreitol (DTT) and sodium dodecyl sulfate (SDS) loading buffer was loaded onto 10‐ or 15‐well 5%−12% SDS‐polyacrylamide gels and wet transferred onto a polyvinylidene difluoride membrane (Bio‐Rad). Nonspecific proteins on the membrane were blocked with either 5% milk, 5% BSA, or 5% milk with 1% BSA in TBST (Tris‐buffered saline, 0.1% Tween 20) at room temperature for 1 h. Then, the blots were probed with primary antibodies (listed in Table S2 ), which were left while rocking at 4°C overnight, followed by incubation with the secondary rabbit or mouse antibody conjugated to horseradish peroxide. The immunoblots were then exposed to Radiance Plus chemiluminescent substrate (Azure Biosystems) and imaged using iBright Imaging Systems (ThermoFisher Scientific) and β‐actin (ACTB) served as the loading control. Blots were quantified using NIH's ImageJ densitometric analysis, with normalization to ACTB.
3
Protein Extraction and Western Blot
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Cells were lysed with RIPA buffer supplemented with protease and phosphatase inhibitor. Extracted proteins were quantified using Pierce BCA assay kit (ThermoFisher Scientific). In all, 40 µg of total protein from each sample were subjected to SDS polyacrylamide gel electrophoresis (PAGE) and electrophoretic transfer to 0.2 µm polyvinylidene difluoride (PVDF) membrane (Bio-Rad) using the Mini Trans-Blot cell (Bio-Rad) at 30 V, 100 mA, 4 °C overnight. The transfer buffer comprises 25 mM Tris, pH 8.3, 192 mM glycine, with 0.03% SDS. The membrane was subsequently blocked in 5% skimmed milk (Nacalai) for total protein immunoblot or 5% BSA (Sigma) for phospho-protein immunoblot. Blots were then incubated with primary antibodies (1:200 for Santa Cruz antibodies and 1:1000 for other antibodies) in appropriate blocking solution at 4 °C overnight, followed by 1 h incubation with horseradish peroxidase conjugated secondary antibody (1:5000) at room temperature. After copious washes with PBS-T (PBS supplemented with 0.1% Tween 20), antibody detection was performed using Radiance plus chemiluminescent substrate (azurebiosystems) and visualized by ChemiDoc MP system (Bio-Rad).
4
Characterizing Amyloid-β Aggregation
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The size of the resulting Aβ preparation after performing each protocol was characterized using SDS-PAGE and western blotting. 120 ng of proteins were separated by electrophoresis in a 12% gel and transferred to a nitrocellulose blotting membrane. After blocking, the membranes were incubated overnight at 4 °C with anti-Aβ primary antibody (6E10 Biolegend, SIG-39320, 1:1000) following incubation with goat anti-mouse secondary antibody (Thermo Scientific, 1:1000) during 1 h. The membranes were subsequently washed and placed in the Radiance Plus Chemiluminescent Substrate (Azure Biosystems, Dublin, CA, USA) and visualized using the Azure c600 gel imaging system (Azure Biosystems).
5
Western Blot Analysis of Protein Expression
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Following SDS-PAGE using 12% gels, proteins were transferred to a nitrocellulose membrane using standard molecular biology procedures. Primary antibodies used were against cystatin C (Abcam, Cambridge, UK; ab109508), GAPDH (Abcam, Cambridge, UK; ab8245), CYB5B (Thermo Scientific, Waltham, MA, USA; PA5-52482), TOM20 (Cell Signaling Technology, Danvers, MA, USA; 42406), TSPO (Abcam, Cambridge, UK; ab109497), IRAP (also known as leucyl-cystinyl aminopeptidase; Cell Signaling Technology, Danvers, MA, USA; 6918), and α-tubulin (Abcam, Cambridge, UK; ab4074). After blocking in 5% milk in TBS supplemented with 0.1% Tween-20 (TBST) and probing with primary antibody, membranes were washed three times with TBST and incubated with secondary antibody conjugated to HRP (Sigma, St. Louis, MO, USA; A0545 or A9044), and then washed for an additional three times with TBST and developed using either Radiance Plus chemiluminescent substrate (Azure Biosystems, Dublin, CA, USA) or SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo scientific, Waltham, MA, USA). Blots were imaged using a ChemiDoc XRS+ imaging system (Bio-Rad, Hercules, CA, USA). Band intensity was assessed via densitometry and normalized against indicated housekeeping protein, and was used as a semi-quantitative estimate of protein concentrations.
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