Alexa fluor 647 conjugated anti ha antibody

Manufactured by BioLegend

Alexa Fluor 647-conjugated anti-HA antibody is a laboratory reagent that binds to the hemagglutinin (HA) epitope tag. The antibody is conjugated to the Alexa Fluor 647 fluorescent dye, which can be detected using appropriate instrumentation.

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Lab products found in correlation

Quikchange lightning mutagenesis kit, by Agilent Technologies (1 mentions) Alexa fluor 488 conjugated streptavidin, by BioLegend (1 mentions) Accuri c6 flow cytometer, by BD (1 mentions) Facsdiva software, by BD (1 mentions) Facscanto, by BD (1 mentions) Ha tag antibody, by BioLegend (1 mentions) Accuri c6 plus, by BD (1 mentions)

Close spelling variants of this product

Alexa Fluor 647–conjugated antibody Alexa Fluor 647 Anti-HA antibody Alexa 647 Anti-HA

3 protocols using alexa fluor 647 conjugated anti ha antibody

Characterization of Polyreactive Nanobody Binding

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Mutations in D06, E10’, and AT118i4h32 were introduced using the Quikchange Lightning mutagenesis kit (Agilent), and yeast were transformed using a standard transformation protocol. Polyreactive nanobody panel and mutant yeast were grown in -Trp + Glu media for two days at 30 °C and induced in -Trp + Gal media at 25 °C for two days. 1 × 10⁶ yeast were washed with DDM selection buffer, and were stained with a 1:10 dilution of either insect cell PSR reagent or Expi cell PSR reagent for 30 min at 4 °C with shaking. Following incubation with PSR reagent, yeast were washed with DDM selection buffer and were stained with a 1:100 dilution of Alexafluor-647 conjugated anti-HA antibody and 1:100 dilution of Alexafluor-488 conjugated streptavidin (Biolegend) for 15 min at 4 °C with shaking. Cells were washed once more with DDM selection buffer and analytical staining was performed using a BD Accuri C6 flow cytometer.

Harvey E.P., Shin J.E., Skiba M.A., Nemeth G.R., Hurley J.D., Wellner A., Shaw A.Y., Miranda V.G., Min J.K., Liu C.C., Marks D.S, & Kruse A.C. (2022). An in silico method to assess antibody fragment polyreactivity. Nature Communications, 13, 7554.

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Quantification of PD-L1 Expression in RRV-infected Cells

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EMT6 cells maximally infected with RRV-scFv-HF-PDL1 (HA-tagged scFv-PD-L1) or RRV-GFP at 90% confluency were treated with recombinant human IFNγ at 250 IU/mL for 24 hours to upregulate PD-L1 expression on the cell surface. One million of IFNγ-treated EMT6 cells maximally infected with RRVscFv-HF-PDL1 or RRV-GFP at indicated ratios were split into 2 sets. One set of cells was stained with Alexa Fluor 647-conjugated anti-HA antibody (BioLegend, # 682404) and the second set of cells was stained with PE-conjugated anti-human PD-L1 antibody (ebioscience, # 12-5983). HA-positive, PD-L1-positive, and GFP-positive cell populations were measured by flow cytometric analysis (FACS Canto, BD Biosciences). HA-positive vs GFP positive cell populations and PD-L1-positive vs GFP-positive cell population were calculated by 2-color compensation for proper gating (BD FACSDiva software, BD Biosciences). Cytometry data analysis was performed using the FlowJo v10 software (FlowJo LCC).

Mitchell L.A., Yagiz K., Hofacre A., Viaud S., Munday A.W., Espinoza F.L., Mendoza D., Rodriguez-Aguirre M.E., Bergqvist S., Haghighi A., Miner M.V., Accomando W.P., Burrascano C., Gammon D., Gruber H.E., Jolly D.J, & Lin A.H. (2019). PD-L1 checkpoint blockade delivered by retroviral replicating vector confers anti-tumor efficacy in murine tumor models. Oncotarget, 10(23), 2252-2269.

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Measuring Surface Expression of NCC Variants

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The surface expression levels of NCC and its variants were analyzed by staining cells using anti-hemagglutinin (HA) tag antibody (catalog number 682404; BioLegend). An HA epitope with flanking “SGSGG” sequence on both sides was inserted into extracellular loop 4 of NCC between residues 414 and 415. The insertion of the HA epitope does not affect the transport activity or biochemical behavior of NCC, as evaluated by a radioactive iodide uptake assay and fluorescence-detection size-exclusion chromatography (FSEC) analysis, respectively. HEK293S cells were cotransfected with HA-tagged NCC variants and a yellow fluorescent protein (YFP), with the latter serving as a marker for successful transfection. After 24 h, cells were harvested and washed with ambient phosphate buffered saline (PBS) once. 0.5 × 10⁶ cells were stained by incubation in PBS with 4 μg/mL Alexa Fluor 647-conjugated anti-HA antibody (catalog number 682404; BioLegend) for 30 min at room temperature. Cells were then washed with ice-cold PBS (x 3 times) and loaded onto the flow cytometer (BD Accuri C6 Plus) for analysis (Supplementary Fig. 2). Cells expressing YFP (FITC positive) were used for measuring surface expression, and surface-expression levels of NCC variants were quantified by the mean fluorescence intensities of Alexa 647.

Fan M., Zhang J., Lee C.L., Zhang J, & Feng L. (2023). Structure and thiazide inhibition mechanism of human Na–Cl cotransporter. Nature, 614(7949), 788-793.

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