Perez-Perri, J.I., Dengler, V.L., Audetat, K.A., Pandey, A., Bonner, E.A., Urh, M., Mendez, J., Daniels, D.L., Wappner, P., Galbraith M.D. and Espinosa, J.M.
Notes: HaloTag® Pull-Down Assay
HEK293T (12 × 106 cells) were plated and grown to 70–80% confluence (approximately 18 hours). The cells were then transfected (using FuGENE® HD Transfection Reagent [Cat.# E2311]) with either 30µg of HaloTag(HT)-HIF1A or HT-alone control vector (vectors available by custom order from Promega Custom Assay Services). Clarified lysates from both HT-HIF1A and HT-alone control cells were prepared and incubated with HaloLink™ Resin (HaloTag® Mammalian Pull-Down System [Cat.# G6500, G6504]). Proteins were digested with trypsin, and digestion was quenched with formic acid. Digested peptides were analyzed by mass spectrometry.
HCT116 and HEK293 cells (8 ×105) were plated in each well of a 6-well plate and co-transfected with one of three acceptors: HT-Pontin, HT-Reptin or HT-TIP60, in combination with the HIF1A-NanoLuc(NL) donor. The following NanoBRET pairs used are available by custom order from Promega Custom Assay Services: HIF1α-NLuc + HT-TIP60, HIF1α-NLuc + HT-Pontin or HIF1α-NLuc + HT-Reptin. (4718)