For Co-immunoprecipitation, HEK293 cells were transfected with PEI in 60 mm tissue culture dishes, and 48 hours post transfection cells were lysed in NP-40 lysis buffer (50 mM Tris-HCl pH 7.5, 0.5% Nonidet p40 (US Biologicals), 150 mM NaCl, 10 mM EDTA, supplemented with 1% HALT protease inhibitor cocktail (Thermo Scientific). Cell lysates were cleared by high speed centrifugation. Immunoprecipitation was performed utilizing anti-FLAG M2 affinity gel or anti-HA conjugated beads (Sigma-Aldrich). Precipitates were washed 3 times in lysis buffer, boiled, and analyzed by Western Blotting.
Anti ha conjugated beads
Manufactured by Merck Group
Anti-HA conjugated beads are a laboratory product designed for the detection and purification of proteins tagged with the hemagglutinin (HA) epitope. These beads are coated with antibodies specific to the HA tag, allowing for the efficient capture and isolation of HA-tagged proteins from complex samples.
Automatically generated - may contain errors
2 protocols using anti ha conjugated beads
1
Co-immunoprecipitation of Proteins in HEK293 Cells
2
CstF-50 Modulates Histone Ubiquitination
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Autoubiquitination reactions were conducted as described previously (6 (link)). The reactions were performed with 1 μg of His-Ub, 0.25 μg of UbcH5c, 20 ng of ΔBRCA1/BARD1 heterodimer, and increasing amounts of GST–CstF-50. Alternatively, 1 μg of commercially available histones (New England BioLabs) or 100 ng of purified RNAP IIO (26 (link)) was added to the ubiquitination reaction mixtures, and conditions were as described previously (29 (link)). The HeLa cells were transfected with HA-Ub and FLAG-histone expression vectors, together with either control or CstF-50 siRNA as indicated previously. Whole-cell extracts were prepared, followed by sonication, and immunoprecipitated with either anti-HA-conjugated beads (Sigma) or anti-FLAG M2 magnetic beads (Sigma) following the manufacturer's instructions. Equivalent amounts of pellets and supernatants were analyzed by immunoblotting.
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