N2A cells were cultured in DMEM medium with high glucose, FBS and PenStrep. These cells were transfected with expression constructs at 60–70% confluency with Transfectin reagent (BioRad, 1703352). Protein lysates were isolated after 48 hours and processed for immunoprecipitation using α-Flag antibody (Sigma, F3165). Dynabeads were used for the IP and proteins were eluted in Lämmli-buffer containing 2-mercaptoethanol, boiled for 10′. Protein samples were separated using 12% SDS-poly-acrylamide gels and transferred to Nitrocellulose membranes (Protan, GE). Primary antibody α-HA antibody (Cell Signalling, 3724) was incubated with the membrane overnight at 4 °C. Secondary antibody horse radish peroxidase conjugated α-rabbit-Ig (Jackson Immunoresearch Labs, 711-035-152) incubation was performed for 1h at RT. Detection was done by chemiluminescence (ECL, GE Healthcare).
α ha antibody
Manufactured by Cell Signaling Technology
The α-HA antibody is a laboratory tool used for detecting and identifying proteins that have been tagged with the HA (hemagglutinin) epitope. The antibody specifically binds to the HA tag, allowing researchers to track and study the expression and localization of HA-tagged proteins in cells and tissues.
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Lab products found in correlation
2 protocols using α ha antibody
1
Immunoprecipitation and Western Blot Analysis
2
Duolink Immunofluorescence Assay for mBim
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For PLA, the Duolink in situ kit (OLINK) was used. Cells were seeded on coverslips in 24-well cell culture plates. After induction of additional 3xHA-tagged mBimEL, cells were washed, fixed, permeabilized with 0.1% Triton X-100/PBS, and blocked with 5% BSA/PBS. Staining was done with α-V5 antibody (1:400; Invitrogen, no. r960-25) and α-HA antibody (1:1500; Cell Signaling, no. C29F4) in 5% BSA/0.5% saponin/PBS. Incubation with conjugated secondary antibodies and detection were performed according to the manufacturer's instructions. Cells were analyzed with the appropriate filters in a Keyence BZ-9000 fluorescence microscope.
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