At 48 h post-transfection, cells were harvested and lysed on ice in RIPA buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.1% SDS, and 0.5% Na-deoxycholate] (Rho et al., 2022 (link)). After centrifugation at 13,000 rpm, the supernatant was incubated with the appropriate antibodies [anti-His (H-3) and anti-E7 (N-19); Santa Cruz Biotechnology, Dallas, TX, USA] overnight at 4°C and then with protein A/G beads (Upstate Biotechnology, Lake Placid, NY, USA) for 3 h. The beads were washed three times with buffer containing 50 mM Tris-HCl (pH 8.0), 1% NP-40, and 2 mM EDTA. Immunoprecipitates were suspended in Laemmli sample buffer and boiled for 5 min. For the immunoprecipitation of endogenous proteins, cells were treated with 5 mM DTBP (dimethyl 3,3’-dithiobispropionimidate-2HCl); Pierce Biotechnology, Waltham, MA, USA) at 4°C for 30 min before harvesting. The cross-linking reaction was stopped by the addition of a wash buffer containing 150 mM NaCl and 100 mM Tris-HCl (pH 8.0). Subsequent procedures were the same as those described above.
Anti his h 3
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-His (H-3) is a monoclonal antibody that recognizes the histidine tag (His-tag) sequence. This antibody is commonly used to detect and purify recombinant proteins that have been engineered to contain a His-tag.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using anti his h 3
1
Immunoprecipitation of Proteins and Cross-linking
2
Characterizing EZH2-VAV1 Protein Interaction
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EZH2-HIS 201-252 and EZH2 with individual mutations were immobilized on the His-Pur Cobalt resin and allowed to interact overnight with GST-VAV1-172 or GST alone (control) in PBS buffer pH 7.4. Following this, the beads were extensively washed and subject to SDS-PAGE and Western blot analysis using anti-His (h-3, Santa Cruz) and anti-GST (B-14, Santa Cruz) antibodies from Santa Cruz to analyze the EZH2-VAV complex. To determine the interaction between full-length EZH2 and VAV, VAV1 protein was immunoprecipitated from the cytosolic fraction of Jurkat cells with α-VAV1 antibody from Santa Cruz (C-14) and immobilized on Protein G Sepharose. Similarly, Flag-tagged EZH2 WT or EZH2 with individual mutations were immunoprecipitated from HEK-293 cells with α-Flag antibody from Sigma (M2) and Protein G Sepharose beads. They were then eluted from the beads by using a competing FLAG peptide. Equal quantities of the WT EZH2 or EZH2 with individual mutations were incubated overnight with VAV1 in a BC-100 buffer. The beads were then extensively washed and analysed by Western blotting with α-EZH2 antibodies from Santa Cruz (N-20) and VAV-specific antibodies.
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