Briefly, the iAT2s were lysed and the virus inactivated by re-suspending the cell pellets in 100 μL of urea lysis buffer (9 M urea, 20 mM HEPES pH 8.0) followed by heating to 100°C for 10 min. Twenty five micrograms of protein lysate was run on a 12% SDS-PAGE Stain-Free gel (Bio-Rad). Stain-free gels were imaged using a ChemiDoc (BioRad) to ensure equal loading before transferring to Nitrocellulose using Turbo TransBlot. Membranes were blocked in 5% milk, 0.02% sodium azide in TBST at room temperature for 1 h. Primary antibodies were incubated overnight at 4°C while rocking in 5% normal horse serum in TBS. Blots were washed 3 times in TBST at room temperature. Secondary antibodies (Immun-Star HRP, BioRad) were incubated with membranes for 1 h at room temperature in 5% normal horse serum in TBS before washing 3 times in TBST. Chemiluminescent detection was performed using Pierce PicoPlus ECL detection reagent and imaged using BioRad ChemiDoc.
Immun star hrp
Manufactured by Bio-Rad
Sourced in United States, China
The Immun-Star HRP is a chemiluminescent detection reagent used for the quantitative or qualitative detection of proteins in Western blotting applications. It is a horseradish peroxidase (HRP)-based system that produces a luminescent signal upon reaction with the HRP enzyme, allowing for the visualization of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc, by Bio-Rad (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Nrf2 antibody, by Abcam (1 mentions)
Multi gauge software, by Fujifilm (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
Goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
High performance chemiluminescence film, by GE Healthcare (1 mentions)
Non fat dry milk, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Anti lamin a c, by Santa Cruz Biotechnology (1 mentions)
Anti pparγ, by Santa Cruz Biotechnology (1 mentions)
Monoclonal anti β actin, by Merck Group (1 mentions)
Anti cox 2, by Santa Cruz Biotechnology (1 mentions)
Anti nf κb p65, by Santa Cruz Biotechnology (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Anti iκbα, by Santa Cruz Biotechnology (1 mentions)
Protein assay kit 2, by Bio-Rad (1 mentions)
Goat anti mouse or goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
7 protocols using immun star hrp
1
Western Blot Analysis of Cell Lysates
2
Western Blot Analysis of Nrf2 and HO-1 in Kidney and Heart
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Kidney cortex and heart were homogenized in cell lysis buffer (Cell Signaling, Danvers, MA) and Western blotting was performed as described previously19 (link). Briefly, samples were subjected to SDS-PAGE and transferred onto a nitrocellulose membrane. After overnight incubation with primary antibody at 4 °C, membrane was incubated with secondary antibody linked with horseradish peroxidase for 1 h at RT. Signal was detected by Immun Star HRP (Bio-Rad, Hercules, CA). The density of each band was determined using Multi Gauge software (Fuji film, Tokyo, Japan) and expressed as a value relative to the density of the corresponding band of the GAPDH. As primary antibodies, Nrf2 antibody (abcam, Cambridge, UK) and heme oxygenase-1 (HO-1) antibody (Enzo Life Sciences, NY, USA) were used. Full-length blots are shown in Supplemental Fig. 1 .
3
SLC38A6 Protein Detection via Western Blot
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Western blot analysis was performed on Immobilon-P PVDF membrane (Millipore) from the 2D gel (described above) in order to detect SLC38A6 protein in cellular fractions. The proteins were pre-blocked for 1 h in blocking buffer (5% nonfat dry milk (Bio-Rad) diluted in 1.5 M NaCl, 0.1 M Tris, 0.05% Tween 20, pH 8.0). Then, the membrane was hybridized with the primary antibody against SLC38A6 (diluted 1∶200) overnight at 4°C. After washes in water, the membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary antibody (diluted 1∶10000, goat anti-rabbit, Invitrogen) followed by detection with the enhanced chemi-luminescent (ECL) method. The membranes were incubated for 3 min in a 1∶1 mixture of luminol/enhancer and peroxidase buffer solutions (Immun-Star HRP, Bio-Rad) and developed on high performance chemi-luminescence film (GE Healthcare).
4
Western Blotting of Cardiac Proteins
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The left ventricular wall tissue was homogenized in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100 and 1 mM PMSF) supplemented with Protease Inhibitor Cocktail (Roche). The supernatant was collected by high-speed centrifugation (12,000 × g) at 4 °C, using BCA method to measure concentration, and mixed with 4× SDS loading buffer (150 mM Tris-HCl, pH 6.8, 12% SDS, 30% glycerol, 0.02% bromophenol blue, 6% 2-mercaptoethanol). The samples were boiled for 10 min. Forty micrograms of protein from each sample was separated using 8%–15% gels by SDS-PAGE and transferred to a nitrocellulose membrane. After blocking with 5% nonfat milk in PBS (pH 7.4), the membranes were incubated with primary antibodies against at 4 °C overnight and visualized by chemiluminescent HRP substrate (Immun-Star HRP, BIO-RAD). Images were quantified using densitometric measurements by Clinx chemi analysis software (Clinx Science Instruments, Shanghai, China, Version 2.1.1.8).
5
Western Blot Analysis of Protein Markers
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Protein concentrations in the cell, nuclei, and cytoplasmic extracts were measured using the Protein Assay Kit 2 according to the manufacturer's instructions (Bio-Rad Laboratories, Hercules, CA, USA). Thirty micrograms of proteins from the cell or nuclei fractions were separated by SDS-polyacrylamide gel electrophoresis using 10% polyacrylamide gel. Proteins were then transferred onto polyvinylidene difluoride membranes (Immobilon-P, Millipore, MA, USA). The membranes were blocked overnight using 5% nonfat milk in 50 mM Tris/150 mM HCl (pH 7.5) containing 0.1% Tween 20 (TBS/Tween). After three 5-min rinses with TBS/Tween, membranes were probed with polyclonal anti-PPARγ, anti-COX-2, anti-p65 NF-κB, or anti-IκBα (Santa Cruz Biotechnology, Heidelberg, Germany) for 1 h at room temperature. After three 5-min rinses with TBS/Tween, horseradish peroxidase- (HRP-) conjugated secondary antibodies goat anti-rabbit or goat anti-mouse IgG (Santa Cruz Biotechnology) were applied for 1 h at room temperature. Protein bands were visualized using a chemiluminescence detection system (Immun-Star HRP; Bio-Rad Laboratories, Hercules, CA, USA). To normalize protein signals, stripped PVDF membranes were reprobed with monoclonal anti-β-actin (Sigma-Aldrich) for cytosolic and cell fractions or with polyclonal anti-lamin A/C (Santa Cruz Biotechnology).
6
Western Blot Protein Extraction Protocol
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Protein extracts were prepared from mouse tissues using RIPA lysis buffer (150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted March 30, 2020. ; https://doi.org/10.1101/2020.03.29.015024 doi: bioRxiv preprint and 50mM Tris, pH=8.0). Protein extracts were denatured with 2X loading buffer (4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue, 0.125M Tris-HCl, pH=6.8) at 95°C for 10 min and run on a 10% SDS-PAGE, followed by transfer to PVDF membrane. The membrane was probed with primary antibodies (Supplementary Table S7) followed by secondary antibody treatment at 1:10000 dilutions (anti-mouse HRP and anti-rabbit HRP, abbkine). Immun-Star™ HRP (1705040, BIO-RAD) was used for chemiluminescence detection and photographed by ChemiDoc XRS+ system (BIO-RAD).
The copyright holder for this preprint this version posted March 30, 2020. ; https://doi.org/10.1101/2020.03.29.015024 doi: bioRxiv preprint and 50mM Tris, pH=8.0). Protein extracts were denatured with 2X loading buffer (4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue, 0.125M Tris-HCl, pH=6.8) at 95°C for 10 min and run on a 10% SDS-PAGE, followed by transfer to PVDF membrane. The membrane was probed with primary antibodies (Supplementary Table S7) followed by secondary antibody treatment at 1:10000 dilutions (anti-mouse HRP and anti-rabbit HRP, abbkine). Immun-Star™ HRP (1705040, BIO-RAD) was used for chemiluminescence detection and photographed by ChemiDoc XRS+ system (BIO-RAD).
7
Protein Extraction and Immunoblotting Protocol
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Cells or sEVs were lysed in RIPA lysis buffer mixing with protease phosphatase inhibitor (Beyotime Biotechnology, Jiangsu, China). The lysis system was incubated on ice for 30 mins, and ultrasonic lysed every 10 mins. Subsequently, the supernatant was carefully removed and transferred into a new microfuge tube after centrifugation at 13000 g for 15mins. The BCA protein assay kit (Beyotime Biotechnology, Jiangsu, China) was used to detect the concentration of proteins according manufacturer's instructions. Each sample (50 μg) was diluted in loading buffer (Beyotime Biotechnology, Jiangsu, China) and subjected to a standard SDS-PAGE followed by transferred onto polyvinylidene uoride membranes (ImmobilonTM-PSQ Membranes, Sigma-Aldrich, China). After blocking in 5% skim milk and washing in Tris Buffered Saline Tween buffer, proteins were detected using the following antibodies: Histone 3 (bs-17422R) at a 1:1000 dilution, CD81 (bs-2489R) at a 1:1000 dilution, TSG101 (bs-1365R) at a 1:1000 dilution, LaminA/C (bs-1839R) at a 1:1000 dilution, and β-actin (bs-0061R) at a 1:2000 dilution. Corresponding secondary antibodies against primary antibodies were used by an hour of incubation (1:2000) . Blots against β-actin served as loading control. Super ECL Plus (Bioground, Chongqin, China) and Immun-Star HRP (BioRad) were used to detect chemiluminescent signals.
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