Equal concentrations of media were mixed with 4X loading buffer (Invitrogen, MA, USA) and boiled at 100°C for 5 min. The SDS-PAGE was performed in 4–20% gradient gel (Bio-Rad, CA, USA). We used SimplyBlue SafeStain (Invitrogen, MA, USA) to visualize the protein band in SDS-PAGE gel. The gel image was captured using the ChemiDoc Imaging System (Bio-Rad, CA, USA). For western blot, equal concentrations of cell lysate and media proteins were transfer into 0.45 μm PVDF membrane (Millipore, MA, USA). Further, blocked with 5% skimmed milk powder at room temperature for 1 h, the membrane was incubated with the target primary antibody with desired dilution (anti-ACE-2, 1:1000 (Cell signaling technology, MA, USA); TMPRSS2, 1:1000 (Santa Cruz Biotechnology, TX, USA); anti-SARS Spike protein 0.05 μg/ml, (Novus Biologicals, CO, USA) at 4°C for overnight. For loading, control membranes were probed with anti-β-actin and anti-GAPDH antibodies (1:5000, Cell signaling technology, MA, USA). The membrane was washed with 1X TBST (3 X 5 min) and then incubated with respective secondary antibodies (Horse anti-mice 1:2000; Horse anti-rabbit 1:2000, (Cell signaling technology, MA, USA) and developed by using ECL chemiluminescent reagent (Bio-Rad, CA, USA).
Anti ace2
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-ACE2 is a laboratory reagent that recognizes and binds to the angiotensin-converting enzyme 2 (ACE2) protein. ACE2 is a key receptor for the SARS-CoV-2 virus, which causes COVID-19. This product can be used to detect and study the ACE2 protein in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Gradient gel, by Bio-Rad (1 mentions)
Ecl chemiluminescent reagent, by Bio-Rad (1 mentions)
Loading buffer, by Thermo Fisher Scientific (1 mentions)
Tmprss2, by Santa Cruz Biotechnology (1 mentions)
Anti β actin and anti gapdh antibodies, by Cell Signaling Technology (1 mentions)
0.45 μm pvdf membrane, by Merck Group (1 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Anti tmprss2, by Abcam (1 mentions)
Horseradish peroxidase conjugated secondary anti mouse or anti rabbit antibodies, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti stat3, by Cell Signaling Technology (1 mentions)
Actin, by Cell Signaling Technology (1 mentions)
Ab54481, by Abcam (1 mentions)
Ab110413, by Abcam (1 mentions)
Ab10983, by Abcam (1 mentions)
Anti pka, by Cell Signaling Technology (1 mentions)
4 protocols using anti ace2
1
SDS-PAGE and Western Blot Analysis
2
Western Blot Analysis of Cellular Proteins
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The treated cellular protein extracts were prepared as described previously (Lin et al., 2017 (link); Lee et al., 2018 (link)). In brief, equal amounts of protein were separated on an 8–10% SDS-PAGE gel and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat dried milk for 30 min and then incubated with primary antibodies for 6–12 h at room temperature. The following primary antibodies were used: anti-ACE2 (1:1,000; Cell Signaling), anti-STAT3 (1:1,000; Cell Signaling), anti-TMPRSS2 (1:1,000; Abcam), anti-β-actin (1:10,000; Santa Cruz), and anti-GAPDH (1:10,000; Santa Cruz) antibodies. All the primary and secondary antibodies were diluted with 1% nonfat dried milk in Tris-buffered saline with 0.1% Tween 20 detergent. The membranes were washed using 0.1% Tris-buffered saline with Tween-20 and incubated in horseradish peroxidase–conjugated secondary anti-mouse or anti-rabbit antibodies (Santa Cruz, ratio: 1:5,000) for 1 h at room temperature. The membranes were washed for 1 h at room temperature. Chemiluminescent protein signals were detected by applying the SuperSignal West Pico PLUS chemiluminescent substrate (Pierce, catalog number: 34087).
3
Protein Expression Analysis Protocol
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Tissues were dissolved in RIPA buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, protease and phosphatase inhibitor mixture (Roche Diagnostics)). Protein concentrations were determined using a BCA assay kit (Pierce Diagnostics). Protein was separated by 10% (wt/vol) SDS/PAGE, transferred to a PVDF membrane (Millipore), blocked in 5% (wt/vol) skim milk in TBST (0.02 M Trisbase, 0.14 M Vehicle, 0.1% Tween 20, pH 7.4), and incubated with primary antibodies overnight at 4 °C and then incubated with secondary antibodies conjugated with HRP. The following primary antibodies were used: anti-UCP1 (ab10983, Abcam), anti-PGC1ɑ (ab54481, Abcam), anti-OXPHOS (ab110413, Abcam), anti-Mas1 (AAR-013, Alomone labs), anti-Akt (#9272, cell signaling technology), anti-p-Akt308 (#13038, cell signaling technology), anti-FoxO1 (#2880, cell signaling technology), anti-p-FoxO1 (#84192, cell signaling technology), anti-PKA (#4782, cell signaling technology), anti-p-PKA (#9621, cell signaling technology), anti-ACE2 (#92485, cell signaling technology), and actin (#4970, Cell Signaling Technology). Signals were detected with Super Signal West Pico Chemiluminescent Substrate (Pierce).
4
SARS-CoV-1 S1 Protein Binding to hACE2
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For CSNPs-binding, hACE2-293T cells were incubated with 10 µM peptide for 1 h and then treated and incubated with 5 µM SARS-CoV-1 S1 protein-His Tag (AcroBiosystems, S1N-C52H3-100UG, USA) for 24 h. The hACE2 over-expression was evaluated in hACE2-293T cells through mRNA expression level using the following primers: (Cosmo Genentech, hACE2-F: 5′-TCC ATT GGT CTT CTG TCA CCC G-3′, hACE2-R: 5′-AGA CCA TCC ACC TCC ACT TCT C-3′, Republic of Korea). After three times whshing with PBS, the cells were incubated with primary antibody anti-ACE2 (1:100, Cell signaling, 4355S, USA), anti-CTNNB1 (1:100, Cell signaling, 8480S, USA), and anti-His-Tag (1:100, Santa Cruz, sc-8036, USA) at 4 °C for 2 h in serum-free media. The cells were fixed with 4% paraformaldehyde for 5 min and incubated with donkey anti-mouse IgG conjugated with Alexa Fluor 594 (1:200, Thermo, A21203, USA) and donkey anti-rabbit IgG Alexa Fluor 488 (1:200, Thermo, A21206, USA) at room temperature for 2 h. Before the secondary antibodies were incubated, cells were blocked with a blocking solution (1% PBS containing 1% BSA and 0.1% Tween 20) at room temperature for 1 h. All secondary antibodies were diluted in an appropriate concentration of blocking solution. Nuclei were stained with DAPI-containing mounting solution (Vector, H-1200, USA). The cells were then visualized on an LSM710 (Carl Zeiss) confocal microscope.
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