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Fitc conjugated anti ha antibody

Manufactured by Merck Group

The FITC-conjugated anti-HA antibody is a laboratory reagent used to detect the presence of the hemagglutinin (HA) tag in protein samples. The antibody is conjugated with the fluorescent dye fluorescein isothiocyanate (FITC), which allows for the visualization of HA-tagged proteins.

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7 protocols using fitc conjugated anti ha antibody

1

Analyzing SCN5A Mutant Expression

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HEK293 cells were transiently co-transfected with HA-tagged SCN5A-WT or HA-tagged SCN5A-N406K respectively. After 48 hours of transfection and with or without mexiletine treatment, the transfected HEK293 cells were harvested by incubation with 0.5 mM EDTA-PBS for 10 min at 37°C and washed with RPMI 1640 supplemented with 1 mM EDTA (pH 7.4), 3%FCS, and 0.02% azide (staining medium). The FITC-conjugated anti-HA antibody (Sigma-Aldrich) incubations were performed in staining medium at 4°C and then washed by PBS supplemented with 1 mM EDTA (pH 7.4) and 1% FCS. The stained cells were examined for surface expression with FACSCalibur (BD Biosciences, San Jose, CA).
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2

Immunofluorescence Staining of CIF Proteins

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Cells were adhered to glass coverslips, treated with PEME buffer for 5 min at room temperature, and then fixed with 4% paraformaldehyde for 10 min at room temperature. Cells were washed once with PBS, incubated with the blocking buffer (3% BSA in PBS) for 1 h at room temperature, and then incubated with the primary antibody for 1 h at room temperature. The following primary antibodies were used: anti-CIF1 polyclonal antibody (1:1,000 dilution) (Zhou et al., 2018a (link)), anti-CIF2 polyclonal antibody (1: 1,000 dilution) (Zhang et al., 2019 (link)), anti-Protein A (anti-ProtA) polyclonal antibody (1:400 dilution) (Sigma-Aldrich), and FITC-conjugated anti-HA antibody (1:400 dilution) (Sigma-Aldrich). Cells were washed three times with PBS containing 0.1% Triton X-100. Except for the FITC-conjugated anti-HA antibody, cells were subsequently incubated with Cy3-conjugated anti-rabbit IgG at room temperature for 1 h. The slides were mounted in VectaShield mounting medium (Vector Labs) containing DAPI and examined using an inverted microscope (Model IX71, Olympus) equipped with a cooled CCD camera (model Orca-ER, Hamamatsu) and a PlanApo N 60 × 1.42-NA DIC objective. Images were acquired and processed using the Slidebook5 software (Intelligent Imaging Innovations, Inc).
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3

Super-Resolution Imaging of XMAP215 in Cytoskeleton

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3D-SIM super-resolution microscopy was carried out according to our published procedures [46 (link)]. Briefly, cells expressing XMAP215-3HA were settled on the coverslips, treated with the PEME buffer containing 1% Nonidet P-40 to prepare cytoskeletons, which were then fixed in cold methanol (-20°C) and incubated in blocking buffer (1% BSA in PBS) at room temperature. Cytoskeletons were co-immunostained with FITC-conjugated anti-HA antibody (Sigma-Aldrich) and YL 1/2 antibody, followed by incubating with Cy3-conjugated anti-rat IgG. Cytoskeletons were imaged under the Nikon Super Resolution Microscope n-SIM E instrument (Nikon Instruments Inc., Americas) with a 100× lens equipped with 488 nm and 592 nm lasers. The acquired SIM images were analyzed by the NIS-Elements AR software.
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4

Transient Transfection and FACS Analysis of WT-Q1077del and Mutant Variants

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HEK293 cells were transiently transfected with HA-tagged WT-Q1077del, HA-tagged R1512W/Q1077del, HA-tagged R1512W/Q1077del+MEX, HA-tagged R1512W/Q1077del/H558R, respectively. After 48 hours of transfection with or without mexiletine treatment, the cells were harvested by incubation with 0.5 mM EDTA-PBS for 10 min at 37°C and washed with RPMI 1640 supplemented with 1 mM EDTA (pH 7.4), 3% FCS, and 0.02% azide (staining medium). The FITC-conjugated anti-HA antibody (Sigma-Aldrich) incubations were performed in a staining medium at 4°C and then washed by PBS supplemented with 1 mM EDTA (pH 7.4) and 1% FCS. The stained cells were examined as previously described [20 ] for quantitative plasma membrane expression with FACSCalibur (BD Biosciences, San Jose, CA).
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5

Quantification of Recombinant Receptor Expression

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Flow cytometry after labeling cells with Phycoerythrin-conjugated anti-FLAG antibody (Biolegend, 637310), Alexa 647-conjugated anti-FLAG antibody (R&D, IC8529R), Alexa 488-conjugated anti-Myc antibody (R&D, IC3696G) or FITC-conjugated anti-HA antibody (Sigma-Aldrich, H7411) was used to quantify recombinant receptor expression levels in HEK293T cells, as described15 (link),30 (link),31 (link). At least 10,000 cells/sample were recorded and analyzed with the FlowJo software (Tree Star).
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6

Immunofluorescence Localization of Cellular Proteins

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Cells were attached to the coverslips for 30 min at room temperature, fixed with cold methanol (−20°C) for 30 min, and rehydrated with PBS for 10 min. Cells on the coverslips were blocked with 3% BSA in PBS for 30 min at room temperature, and incubated with anti-CC2D polyclonal antibody (1 : 2000 dilution) [47 (link)], anti-CIF1 polyclonal antibody (1 : 1000 dilution) [22 (link)], anti-CIF2 polyclonal antibody (1 : 1000 dilution) [39 (link)], anti-Protein-A polyclonal antibody (Sigma-Aldrich, 1 : 400 dilution) or FITC-conjugated anti-HA antibody (Sigma-Aldrich, clone HA-7, 1 : 400 dilution) for 1 h at room temperature, or with anti-TbPLK polyclonal antibody (1 : 160 dilution) [17 (link)] for 1 h at 37°C. After the cells on the coverslips were washed three times with PBS, cells were incubated with Cy3-conjugated anti-rabbit IgG (Sigma-Aldrich, 1 : 400 dilution) for 1 h at room temperature. After three washes with PBS, the coverslips were mounted in DAPI-containing VectaShield mounting medium (Vector Laboratories) and examined using an inverted microscope (model IX71, Olympus) equipped with a cooled CCD camera (model Orca-ER, Hamamatsu). Images were acquired and processed with the Slidebook software (Intelligent Imaging Innovations).
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7

Endogenous Tagging of Hypothetical Proteins

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The 30 hypothetical proteins whose transcripts were highly enriched in S phase (Supplementary Table S1), the 21 orphan kinesins and kinetoplastid-specific kinesins (Supplementary Table S2), and the T. brucei Fidgetin homolog (Tb927.5.1870) were each endogenously tagged with a triple HA epitope by the PCR-based gene tagging method (28 (link)) in the 427 cell line. Transfectants were selected with 1 μg/ml puromycin and cloned by limiting dilution in a 96-well plate containing SDM-79 medium supplemented with 20% heat-inactivated fetal bovine serum. Western blotting was carried out with anti-HA antibody (clone# HA-7, Catalog # H3663, Sigma-Aldrich) to confirm the tagging of proteins. Cells were then immunostained with FITC-conjugated anti-HA antibody (1:400 dilution, Clone# HA-7, Catalog# H7411, Sigma-Aldrich) and imaged under an inverted fluorescence microscope.
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