COS-7 cells were transfected with either PRRT2-HA or BAP-HA used as a control; after 24 h cells were harvested in lysis buffer. Monoclonal anti-HA-agarose affinity resin (Sigma Aldrich) was incubated with cell extracts according to manufacturer’s instructions at 4 °C for 4 h. After four washes in lysis buffer, immunocomplexes were incubated at 4 °C overnight with total mouse brain extracts to allow binding of potential interacting proteins. Pulled down proteins were eluted by HA peptide solution (200 µg/mL HA-peptide in lysis buffer), subjected to SDS–PAGE on 10% polyacrylamide gels, and processed for Coomassie staining, Western blotting, and mass spectrometry.
Monoclonal anti ha agarose affinity resin
Manufactured by Merck Group
Monoclonal anti-HA-agarose affinity resin is a laboratory product used for the purification and detection of proteins tagged with the hemagglutinin (HA) epitope. It consists of a monoclonal antibody specific to the HA tag that is covalently linked to agarose beads, providing a solid-phase matrix for affinity-based separation and enrichment of HA-tagged proteins from complex samples.
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Lab products found in correlation
2 protocols using monoclonal anti ha agarose affinity resin
1
Identifying PRRT2 Protein Interactors
2
PRRT2 Interaction with Cav2 Channels
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HEK293 cells were co-transfected with PRRT2-HA and either Cav2.1 or Cav2.2, as described above. Cells were harvested in lysis buffer (150 mM NaCl, 50 mM Tris, 1 mM EDTA and 1% Triton X-100 supplemented with protease inhibitor cocktail) and centrifuged at 10,000 x g for 10 min at 4°C. Kept an aliquot for input, the supernatant was incubated with 50 μL of monoclonal anti-HA-agarose affinity resin (Sigma-Aldrich) according to manufacturer’s instructions for 2 h at 4°C. After extensive washes in lysis buffer and detergent-free lysis buffer, samples were resolved by SDS-PAGE and subjected to western blotting with anti-HA, anti-Cav2.1 and anti Cav2.2 antibodies.
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