We homogenized frozen brain tissues (175 ± 20 mg) in 5% glucose in distilled water in grinding tubes (Bio-Rad, Hercules, CA, USA) adjusted to 10% (wt/vol) by using a TeSeE Precess 48TM homogenizer (Bio-Rad), according to the manufacturer’s instructions. We determined presence of PrPres in transgenic mouse brains by Western blot, using the reagents of the ELISA commercial test TeSeE (Bio-Rad). Based on a previously described protocol (31 (link)), we treated 10–100 μL of 10% wt/vol brain homogenates with proteinase K; the resulting samples were loaded in 12% Bis-Tris Gel (Criterion XT; Bio-Rad). We transferred proteins electrophoretically onto PVDF membranes (Millipore, Billerica, MA, USA), which were blocked overnight with 2% BSA blocking buffer (Sigma-Aldrich, St. Louis, MO, USA). For immunoblotting, we incubated with Sha 31 (44 (link)) monoclonal antibody (mAb) at a concentration of 1 µg/mL to identify the 145-WEDRYYRE-152 epitope of the human PrPC sequence. To detect immunocomplexes, we incubated the membranes for 1 h with horseradish peroxidase conjugated anti-mouse IgG (GE Healthcare Amersham Biosciences, Little Chalfont, UK). Immunoblots were developed with enhanced chemiluminiscence ECL Select (GE Healthcare Amersham Biosciences). Images were captured using the ChemiDoc WRS+ System (Bio-Rad) and processed using Image Lab 5.2.1 software (Bio-Rad).
Ecl select
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Italy, Sweden, Spain
ECL Select is a chemiluminescent substrate solution designed for the detection of protein targets in Western blot analysis. It provides a sensitive and reliable method for visualizing protein bands on a membrane.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (7 mentions)
Hybond p pvdf membrane, by GE Healthcare (6 mentions)
Ecl prime, by GE Healthcare (4 mentions)
Hyperfilm mp, by GE Healthcare (4 mentions)
Las 3000mini luminoimage analyzer, by Fujifilm (4 mentions)
Pvdf membrane, by Bio-Rad (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Odyssey fc, by LI COR (3 mentions)
Bioruptor, by Diagenode (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Complete protease inhibitor cocktail, by Roche (3 mentions)
Image lab 5, by Bio-Rad (2 mentions)
Grinding tubes, by Bio-Rad (2 mentions)
Criterion xt, by Bio-Rad (2 mentions)
Bsa blocking buffer, by Merck Group (2 mentions)
Chemidoc wrs system, by Bio-Rad (2 mentions)
Criterion tris hcl precast gel, by Bio-Rad (2 mentions)
Peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Mcl 1, by Cell Signaling Technology (2 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
77 protocols using ecl select
1
PrP^res Detection in Transgenic Mouse Brains
2
Protein Extraction and Western Blot Analysis
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Cell pellets were resuspended and lysed for 30 min on ice in RIPA lysis buffer supplemented with Protease/Phosphatase Inhibitor (1:100, Thermo Fisher Scientific, Massachusetts, USA). Protein concentration was determined using the BCA Protein Assay Kit (Pierce—Thermo Fisher Scientific, Massachusetts, USA) when necessary. Lysate was denatured by adding 4 × Laemmli Buffer (Biorad, California, USA) supplemented with 10% β-mercapto-ethanol and subsequent heat treatment at 95 °C for 10 min. Proteins were separated by SDS-PAGE. Samples were loaded onto precast gels (Invitrogen) and transferred to polyvinylidene difluoride membranes. The membranes were incubated in PBS-T (5% milk) blocking solution for 2 h at room temperature, followed by incubation with the respective monoclonal Ab, overnight in the dark at 4 °C. Each membrane was then washed three times with PBS-T and incubated with HRP-conjugated anti-rabbit Ab for 30 min at room temperature. Immunoreactive proteins were visualized using ECL Prime and/or ECL Select (GE Healthcare).
3
Western Blot Analysis of Cell Lysates
4
Blue Native-PAGE Immunoblotting Protocol
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Blue native-PAGE was performed using Novex 4–12% gradient gels (Thermo Fisher Scientific, MA, USA). The primary antibodies (C211 and FL-140) were prepared by addition of 5% Coomassie G-250 additive. Blue Native-polyacrylamide gels were then run at 150 V for 2 hours according to the manufacturer’s protocol, and the separated proteins were then transferred onto PVDF membranes (Merck Millipore, Germany). The membranes were then blocked with 5% nonfat milk in PBS containing 0.1% (v/v) Tween 20 for 30 min at RT, and subsequently incubated for 24 hours with plasma samples diluted to 50% in PBS, which exhibited either high or low HA activity in preparatory experiments. After washing with PBST, the membranes were incubated in HRP-labeled anti-human immunoglobulin antibody (DAKO, Denmark) for 3 hours. Immunosignals were visualized using chemiluminescence (ECL Select; GE Healthcare, England).
5
Quantification of Cytokine and Protein Levels
6
Quantitative Analysis of Arc Protein in Hippocampal Subregions
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Mouse hippocampi were quickly removed and one was processed as whole hippocampus and from the other, hippocampal subareas CA1 and DG were dissected as described [69 (link)]. Hippocampal tissue was homogenized on ice in NETN buffer (Tris pH 8.0 50 mM, NaCl 250 mM, EDTA 5 mM, NP40 0.5 %, supplemented with protease inhibitors, Complete Roche). Extracts were cleared by centrifugation (14,000 g, 20 min). Samples (15 μg) were analyzed by SDS-PAGE and immunoblot. PVDF membranes were incubated with anti-Arc (C-7, Santa Cruz) and as loading control, with anti-Erk2 (C-14, Santa Cruz). Secondary antibodies used were HRP-conjugated donkey anti-rabbit or -mouse IgG (Jackson; 1 h, room temperature), and signals were detected with ECL Select (GE Healthcare). Band intensity was quantified with QuantityOne software (BioRad).
7
Decellularized Mouse dECM Proteome Analysis
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Decellularized and recellularized mouse dECM, fresh diaphragm, and hMPCs were solubilized in 1 ml of T‐PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific) and homogenized using TissueLyser (Qiagen, Hilden, Germany), according to manufacturer instructions. Then, equal amounts of protein extracts (25 μg) were resolved by SDS‐PAGE gels (4%–12%, 3%–8%) and transferred to nitrocellulose transfer membrane (Advansta, California, United State). Membranes were blocked with Blotto nonfat dry milk (Santa Cruz, California, United State) for at least 1 hour at RT and then incubated overnight at 4°C under constant shaking with primary antibodies listed in Supporting Information Table S1 . Membranes were next incubated with peroxidase‐labeled secondary antibodies (Supporting Information Table S1 ), visualized using ECL Select, and exposed to Amersham Hyperfilm ECL (GE Healthcare, Milan, Italy).
8
Myc-tag Protein Detection by Western Blot
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3 OD600 units of cells were harvested by centrifugation, resuspended in 100 μl of 1.85 M NaOH containing 7% β-Mercaptoethanol and incubated on ice for 10 min, followed by addition of 100 μl of 50% TCA and incubation on ice for 5 min. After centrifugation at 13,000 g for 10 min, protein pellets were washed twice with 1 M Trizma base (Merck, Darmstadt, Germany) before resuspension in 100 μl 2× SDS-PAGE loading buffer (100 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 4 mM EDTA, 0.2% bromophenol blue, 2% β-Mercaptoethanol). Samples were incubated at 95°C for 10 min, briefly centrifuged and analyzed by SDS-PAGE and immunoblotting using anti-Myc antisera (9E11, Santa Cruz Biotechnology, Dallas, Texas, USA). Immunoreactive signals were detected with a horseradish peroxidase-conjugated secondary antibody (P0447, Dako, Santa Clara, California, USA) reacted with ECL Select (GE Healthcare, Little Chalfont Buckinghamshire, United Kingdom) and images were acquired with a FujiFilm LAS-1000 camera using the Intelligent Dark Box II (FujiFilm, Tokyo, Japan).
9
Western Blot Protein Detection
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Cells were lysed in RIPA buffer (20 mM Tris pH 7.4, 150 mM NaCl, 1% NP40, 0.1% SDS and 0.5% sodium deoxycholate, containing protease inhibitor cocktail cOmplete (Sigma-Aldrich). Protein samples were subjected to 10% SDS-PAGE and transferred to Hybond-ECL (GE Healthcare, Pittsburgh, PA, USA). The blotted membranes were blocked in 2% non-fat milk for 30 min and then incubated with primary antibodies (1:2,000) at 4°C overnight, followed by incubation with secondary antibodies (1:20,000) at room temperature for 1 h, and visualized using ECL Select (GE Healthcare).
10
Western Blot Protein Detection
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Total protein was extracted from the cell pellets using RIPA buffer and separated by SDS-PAGE. Protein was then transferred onto the PDVF membrane, which was blocked in 5% BSA blocking buffer for 2 h at room temperature. Then, the membrane was incubated with primary antibodies overnight at 4 °C followed by secondary antibody incubation for 2 h at room temperature. The membrane was visualized with chemiluminescence (Bio-Rad, USA) using ECL Select as a substrate (GE Healthcare, Germany).
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