The cells were fixed at 48 h post-transfection with 2% paraformaldehyde for 10 min, followed by incubation with 0.1% Triton X-100 for 10 min. After blocking with 5% bovine serum albumin, mouse anti-HA tag antibody (Invitrogen, CA, USA) was added to each well and incubated for 1 h. Following washing for three times with PBS, each well was incubated with FITC-conjugated goat anti-mouse IgG (Abcam, UK) at 37 °C for 1 h. The cells were observed under a Nikon fluorescence microscope (Nikon, Japan).
Mouse anti ha tag antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Mouse anti-HA tag antibody is a laboratory tool used to detect and identify proteins that have been engineered to contain the HA (Hemagglutinin) tag sequence. This antibody specifically binds to the HA tag, allowing for the visualization and tracking of HA-tagged proteins in various experimental procedures.
Automatically generated - may contain errors
3 protocols using mouse anti ha tag antibody
1
Immunofluorescence Staining of HA-Tagged Proteins
2
Immunofluorescence Localization of HA-tagged Proteins
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On day 2 post-transfection, the transfected LMH cells were fixed with 2% paraformaldehyde for 10 min, followed by treatment with 0.1% Triton X-100. Then, the cells were blocked with 5% bovine serum albumin. Following washing, the cells were incubated with mouse anti-HA tag antibody (Invitrogen, Carlsbad, CA, USA) for 1 h. Then, the cells were washed three times with PBS, followed by incubation with FITC-conjugated goat anti-mouse IgG (Abcam, Cambridge, UK) at 37°C for 30 min. An inverted fluorescence microscope (Nikon, Tokyo, Japan) was used to examine the fluorescent signal.
3
Western Blot Analysis of Epitope-Tagged Proteins
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20 μL per lane of epitope-tagged Pmt eluent was loaded into 2 gels to be further probed with anti-S tag or anti-HA antibody. The proteins were resolved on 4–20% gradient Tris–HEPES SDS gels (Pierce Protein Gels, Thermo Fisher Scientific Inc., IL) and transferred to a nylon membrane. S-tagged Pmts were detected with mouse anti-S-tag antibody (1:5000) (Abcam Inc., MA), followed by Sheep anti-Mouse IgG ECL antibody coupled to horseradish peroxidase (1:5000) (GE Healthcare, NJ). HA-tagged proteins were detected with mouse anti-HA-tag antibody (1:5000) (Invitrogen Co., CA), followed by Sheep anti-Mouse IgG ECL antibody coupled to horseradish peroxidase (1:5000) (GE Healthcare, NJ).
For Pmt target protein analyses, an equal volume of 2 × SDS–PAGE sample buffer [27] was added to membrane fractions and heated for 5 min at 95°C. Forty μg solubilized protein were loaded per lane and detected as described above.
For Pmt target protein analyses, an equal volume of 2 × SDS–PAGE sample buffer [27] was added to membrane fractions and heated for 5 min at 95°C. Forty μg solubilized protein were loaded per lane and detected as described above.
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