goat anti human ace2 primary antibody  (R&D Systems)

Journal: PLOS One

Article Title: pET28g: A Golden Gate-compatible pET vector for protein expression in Escherichia coli , validated by production of functional human ACE2

doi: 10.1371/journal.pone.0327341

Figure Lengend Snippet: Protein expression as detected in total soluble protein extracts by western blot with anti-ACE2 antibody (A and B) and Coomassie Brilliant Blue staining (C and D) . A and C, soluble protein extracts of bacteria transformed with pET28a plasmid expressing ACE2 with a 6x-His tag at the N-terminus; B and D, soluble protein extracts of bacteria transformed with pET28g plasmids expressing ACE2 with an 8 × His-Gb1 tag (Gb1) or an MBP tag (MBP) at the N-terminus. NI – Soluble protein extract from bacteria before protein expression induction; I4 – Soluble protein extract from bacteria four hours after protein expression induction; M – Molecular weight marker (MB09002, NZYTech).

Article Snippet: Detection of ACE2 was performed using an anti-ACE2 primary antibody (1:2,000 dilution, AAF933 from R&D Systems) and HRP-conjugated anti-goat secondary antibody (1:20,000 dilution, sc-2020 from Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Staining, Bacteria, Transformation Assay, Plasmid Preparation, Molecular Weight, Marker

Journal: PLOS One

Article Title: pET28g: A Golden Gate-compatible pET vector for protein expression in Escherichia coli , validated by production of functional human ACE2

doi: 10.1371/journal.pone.0327341

Figure Lengend Snippet: × His-Gb1 tag . A) SDS-PAGE analysis of fractions from the reverse HisTrap purification step, performed after TEV protease cleavage to remove the 8 × His-GB1 tag and His-tagged TEV protease. The flowthrough (FT) contains the cleaved (untagged) ACE2, while the elution fraction contains the His-tagged TEV protease and the cleaved His-tag. Washes 1 and 2 (W1, W2) with PBS pH 7.4 ensured complete recovery of untagged ACE2. The figure also shows fractions (a) and (b) collected from size-exclusion chromatography. (B) Size-exclusion chromatography profile of untagged ACE2 purified using a HiLoad 16/600 Superdex™ 200 pg column, where peak (b) corresponds to the untagged ACE2 protein. M – molecular weight marker (MB09002, NZYTech).

Article Snippet: Detection of ACE2 was performed using an anti-ACE2 primary antibody (1:2,000 dilution, AAF933 from R&D Systems) and HRP-conjugated anti-goat secondary antibody (1:20,000 dilution, sc-2020 from Santa Cruz Biotechnology).

Techniques: SDS Page, Purification, Size-exclusion Chromatography, Molecular Weight, Marker

Journal: PLOS One

Article Title: pET28g: A Golden Gate-compatible pET vector for protein expression in Escherichia coli , validated by production of functional human ACE2

doi: 10.1371/journal.pone.0327341

Figure Lengend Snippet: (A) Far-UV CD spectra of ACE2 with a characteristic α-helical profile, indicating a predominantly helical secondary structure. Measurements were performed at a protein concentration of 0.2 mg/mL in PBS, pH 7.4, using a 0.1 cm path length cuvette at 25°C. Baseline correction was applied using the buffer CD spectrum. (B) Rates of Mca-APK(Dnp) substrate cleavage by recombinant ACE2. The rates are given in fluorescence arbitrary units (a.u.) because the unquenched Mca substrate was not available to prepare a calibration curve. For comparison, the rates observed for the control reactions, in the absence of substrate or protein, were plotted. Differences between the rate determined for ACE2 and the controls are statistically significant.

Article Snippet: Detection of ACE2 was performed using an anti-ACE2 primary antibody (1:2,000 dilution, AAF933 from R&D Systems) and HRP-conjugated anti-goat secondary antibody (1:20,000 dilution, sc-2020 from Santa Cruz Biotechnology).

Techniques: Circular Dichroism, Protein Concentration, Recombinant, Fluorescence, Comparison, Control

Journal: Viruses

Article Title: SARS-CoV-2 Modulation of HIV Latency Reversal in a Myeloid Cell Line: Direct and Bystander Effects

doi: 10.3390/v16081310

Figure Lengend Snippet: ( A ) Relative ACE2 expression levels in different U1 and U937 cells ( left ) and representative histograms of median fluorescence intensity (MFI) for each condition ( right ). Among U1, different conditions were evaluated, such as US: unstimulated U1; cfv: U1 exposed to SARS-CoV-2 cell-free; U937: unstimulated U937. U1 exposed to conditioned media (CM) obtained from non-infected MDM (Ni), MDM exposed to SARS-CoV-2 (Wh), MDM polarized (M1, or M2). PMA (Phorbol 12-myristate 13-acetate): positive control for reversal on both HIV-latently infected cell models. Data show the mean ± SEM, and statistical significance was calculated by one-way ANOVA. ( B ) Kinetics of the SARS-CoV-2 (Wh variant, MOI: 0.1) replication in myeloid (U1, U937) cells measured using RT-qPCR targeted to N and ORF1a genes in culture supernatant, as described in M&M. T0: supernatant obtained after three cell washes. 72 h: 3 days post-infection. Data are expressed as mean ± SD obtained from 4 independent experiments. (* p < 0.05; *** p < 0.001).

Article Snippet: A rabbit anti-human ACE2 primary polyclonal Ab (ab272690, Abcam, Cambridge, UK) and goat anti-rabbit IgG secondary (PE) (Abcam, UK) were used for ACE2 quantification.

Techniques: Expressing, Fluorescence, Infection, Positive Control, Variant Assay, Quantitative RT-PCR