Human embryonic kidney (HEK) 293 or S2 cells were transfected with Effectene for 2 to 5 days. Cell lysis and immunoprecipitation with GFP-Trap_A (ChromoTek GmbH), anti-Flag M2 affinity gel (Sigma-Aldrich), or anti–hemagglutinin (HA)–agarose (Sigma-Aldrich) were performed according to the manufacturer’s instructions. Input ranged from 0.2 to 0.4% of lysis volume. Samples were electrophoresed on 12.5% SDS–polyacrylamide gels and transferred to nitrocellulose membranes, blocked in 5% skim milk powder in tris-buffered saline overnight, and subsequently incubated with primary and secondary antibodies. Chemiluminescence detection was performed on a VersaDoc imager (Bio-Rad).
Anti hemagglutinin ha agarose
Manufactured by Merck Group
Anti–hemagglutinin (HA)–agarose is a laboratory equipment product used for protein purification. It consists of agarose beads covalently linked to anti-HA antibodies, which can be used to selectively capture and purify HA-tagged proteins from complex samples.
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Lab products found in correlation
Gfp trap a, by Proteintech (1 mentions)
Versadoc imager, by Bio-Rad (1 mentions)
Anti flag m2 affinity gel, by Merck Group (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Anti flag horseradish peroxidase hrp, by Merck Group (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
2 protocols using anti hemagglutinin ha agarose
1
Immunoprecipitation of GFP, Flag, and HA Proteins
2
Immunoprecipitation and Western Blot Analysis
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Cells were lysed in modified radioimmunoprecipitation assay (RIPA) buffer [20 mM tris-Cl (pH 7.5), 2 mM EDTA, 420 mM NaCl, and 1% NP-40]. After centrifugation, the soluble fractions were collected and incubated with anti-hemagglutinin (HA) agarose (Sigma) at 4°C for 2 hours. The precipitates were then washed three times with modified RIPA buffer and boiled for 5 min in SDS loading buffer. Samples were analyzed by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane (Millipore), and probed with anti-FLAG horseradish peroxidase (HRP) (Sigma). Immune complexes were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore). Densitometry analysis of Western blots was performed using the ImageJ software. All immunoprecipitation experiments were performed at least three times with similar findings.
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