HA Tag Alexa Fluor® 488-conjugated Antibody

Detection of HA Tag in HEK293 Human Cell Line Transfected with HA-tagged Proteins by Flow Cytometry.

HEK293 human embryonic kidney cell line transfected with either (A) HA-tagged proteins (filled histogram) or (B) irrelevant transfectants (open histogram) was stained with Mouse Anti-HA Tag Alexa Fluor® 488-conjugated Monoclonal Antibody (Catalog # IC6875G). To facilitate intracellular staining, cells were fixed and permeabilized with FlowX FoxP3 Fixation & Permeabilization Buffer Kit (Catalog # FC012). View our protocol for Staining Intracellular Molecules.

Detection of HA Tag by Western Blot

SARS-CoV-2 VLPs with N-glycan mutations display reduced viral entry.(A) Plasmids encoding for the four structural proteins containing distinct tags or EGFP reporters were either transfected alone or pooled and transfected together into 293Ts. (B) Confocal micrographs show the intracellular colocalization of M, N, E, and Spike in perinuclear compartments that likely includes the ERGIC (arrowhead). (C) Flow cytometry analysis performed with live and “fixed-permeabilized cells” demonstrate cell surface expression of Spike and M proteins. N and E were intracellular. Here, the structural proteins were either expressed alone or altogether: “+” and “−” indicate presence/absence of indicated proteins. (D) All proteins were expressed in VLPs (detected using epitope-tag Abs), but Spike carrying N61Q and N801Q mutations displayed somewhat reduced expression. (E) Luc-VLPs containing firefly luciferase reporter were produced by cotransfecting 293T cells with plasmids encoding for N (R203M mutant), M-IRES-E, Luc-PS9, and Spike constructs. Luc-VLP viral entry into 293T/ACE2 cells was reduced upon implementing many of the N-to-Q glycosylation mutations. “No-spike” VLPs were made using all plasmids except Spike. Mock infection did not contain VLP. Data were normalized to parent-VLP luminescence in each run [both for (E) and (F)]. (F) Luc-VLP with parent-Spike, produced using 293T cells, was deglycosylated using either Endo H or peptide N-glycosidase (PNGase) F. PNGase F reduced viral entry into 293T/ACE2 and Calu-3 cells, with a smaller effect being observed for Endo H. Data are means ± SD for n ≥ 3. *P < 0.05 with respect to all other treatments and †P < 0.05 with respect to parent. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36149962), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of HA Tag by Western Blot

SARS-CoV-2 VLPs with N-glycan mutations display reduced viral entry.(A) Plasmids encoding for the four structural proteins containing distinct tags or EGFP reporters were either transfected alone or pooled and transfected together into 293Ts. (B) Confocal micrographs show the intracellular colocalization of M, N, E, and Spike in perinuclear compartments that likely includes the ERGIC (arrowhead). (C) Flow cytometry analysis performed with live and “fixed-permeabilized cells” demonstrate cell surface expression of Spike and M proteins. N and E were intracellular. Here, the structural proteins were either expressed alone or altogether: “+” and “−” indicate presence/absence of indicated proteins. (D) All proteins were expressed in VLPs (detected using epitope-tag Abs), but Spike carrying N61Q and N801Q mutations displayed somewhat reduced expression. (E) Luc-VLPs containing firefly luciferase reporter were produced by cotransfecting 293T cells with plasmids encoding for N (R203M mutant), M-IRES-E, Luc-PS9, and Spike constructs. Luc-VLP viral entry into 293T/ACE2 cells was reduced upon implementing many of the N-to-Q glycosylation mutations. “No-spike” VLPs were made using all plasmids except Spike. Mock infection did not contain VLP. Data were normalized to parent-VLP luminescence in each run [both for (E) and (F)]. (F) Luc-VLP with parent-Spike, produced using 293T cells, was deglycosylated using either Endo H or peptide N-glycosidase (PNGase) F. PNGase F reduced viral entry into 293T/ACE2 and Calu-3 cells, with a smaller effect being observed for Endo H. Data are means ± SD for n ≥ 3. *P < 0.05 with respect to all other treatments and †P < 0.05 with respect to parent. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36149962), licensed under a CC-BY license. Not internally tested by R&D Systems.