After enriching U251 cell lysate by a lactic probe with a photo-cross linker and a corresponding control probe without lactic modification to find related reader or eraser, the protein enriched were identified by Lc-MS (three replicates)

ABSTRACT: In brief, U251 cells lysate were incubated with 10μM KpKla and 10μM KpKla separately. After incubating for 1h at 37℃, the solutions were irradiated by 365nm UV for 5 min on ice. Then CuSO4, THPTA (tris-hydroxypropyltriazolylmethylamine), TCEP (tris(2-carboxyethyl)phosphine) and biotin azide were added and the mixture was stirred vigorously for 2h. After that, chilled acetone was added and the mixtures were centrifuged at 14000r/min for 15min at 4℃. The supernatant was disposed and the precipitate was washed with chilled methanol. After that, the precipitate was redissolved with 1% SDS in PBS. The mixture was centrifuged again at 14000r/min for 15min to remove the insoluble precipitate. After collecting the supernatant, 100μL of streptavidin agarose beads (Theromal Fisher) were added to each tube. The solution and the beads were incubated overnight at 4 ℃. Then the supernatant was discarded, and the beads were washed with 0.5% SDS in PBS, 0.1% SDS in PBS and PBS three times(shaking for 15 min each time) separately. 1000r/min centrifuge was used to dispose the solvent. Finally, 50μL 1X loading buffer was added to each tube. The supernatant was used for SDS-page after boiling and centrifuge at 14000rpm. After the SDS page and Coomassie staining, the Coomassie stained gel particles were cut and collected into 1.5mL tubes for LC-MS analysis.

ORGANISM(S): Homo Sapiens

SUBMITTER: Quan Hao  

PROVIDER: PXD039731 | iProX | Sun Jan 29 00:00:00 GMT 2023

REPOSITORIES: iProX