T24 cells were plated overnight in T-25 flasks (5×105 cells/flask) and transiently transfected with 10 μg of ACE2-GFP, dACE2-GFP, or empty GFP-vector. After 24 hrs post-transfection, cells were pelleted and lysed with 400 μl of Lysis Buffer provided with the ACE2 activity kit (#K897, BioVision). Keeping the reaction volumes and the amount of ACE2-GFP lysates constant, we added dACE2-GFP lysate in the ratio of 0.25, 0.5 and 1.0 to ACE2-GFP, with differences in volume compensated by lysates from GFP-expressing cells. The lysate mixtures were processed in triplicates using the kit reagents and according to protocol. The carboxypeptidase activity was measured as fluorescence (Ex/Em = 365/410–460 nm) using a Promega GlowMax plate reader for two time points between 30 mins and 2 hrs after adding the corresponding substrate mix. A positive control was provided by the kit. Cell lysates were also analyzed by Western blots with C-terminal anti-ACE2 antibody (Abcam), that detects both ACE2 and dACE2, with GAPDH as a loading control.
C terminal anti ace2 antibody
Manufactured by Abcam
The C-terminal anti-ACE2 antibody is a laboratory research tool used to detect and study the expression of the angiotensin-converting enzyme 2 (ACE2) protein. ACE2 is a receptor for the SARS-CoV-2 virus, which causes COVID-19. This antibody binds to the C-terminal region of the ACE2 protein.
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Lab products found in correlation
2 protocols using c terminal anti ace2 antibody
1
Measuring ACE2 Activity in Transfected Cells
2
Quantifying ACE2 Protease Activity
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T24 cells were transiently transfected with ACE2-GFP, dACE2-GFP, or empty GFP-vector in triplicates. After 24 hrs, cells were pelleted and lysed with 400 μl of Lysis Buffer provided with the ACE2 activity kit (#K897, BioVision). Cell lysates were analyzed by Western blots with C-terminal anti-ACE2 antibody (Abcam), and the amounts of recombinant ACE2-GFP and dACE2-GFP proteins in lysates were quantified by densitometry analysis (Imagelab, BD Biosciences) of Western blots. The amounts of ACE2 and dACE2 lysates to be used in protease assays were determined based on protein quantification. Protein lysates were processed in duplicates using the kit reagents and according to protocol. The activity was measured as fluorescence (Ex/Em = 365/410–460 nm) using Promega GlowMax plate reader for two time points between 30 mins and 2 hrs after adding the corresponding substrate mix and normalizing for baseline level. Positive control was provided by the kit.
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