Mammalian mitochondrial polyphosphate is regulated by the 5-InsP7 synthase IP6K1

ABSTRACT: HEK293T cells were collected and lysed by incubating it in lysis buffer containing 50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM NaF, 1X protease inhibitor cocktail, 1X phosphatase inhibitor cocktail for 45 mins at 4 degree C followed by centrifugation at 8000g for 10 mins at 4 degree C. Cell lysate containing 1 mg protein was added to 100 microlitre each of 100 micromolar PolyP8-agarose, PolyP3-agarose, Pi agarose, and hydroxy agarose. The volume was made up to 1ml using lysis buffer. The beads were kept overnight at 4 degree C for overnight mixing on an end-over-end mixer, washed using lysis buffer, and boiled at 95 degree C for 5 mins in 1X Laemmli buffer. The proteins were run on 12% SDS-Polyacrylamide gels for 20 mins so that the dye front moves to about 0.8 cm in the resolving gel. The gel was stained using the Coomassie blue stain (0.25%) for half an hour. Later, the gel was destained using a destaining solution to remove excess stain. The stained protein band was cut and sent for Mass spectrometry to TAPLIN facility at Harvard University.

INSTRUMENT(S): LTQ Orbitrap Velos Pro

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Rashna Bhandari  

PROVIDER: MSV000098211 | MassIVE | Mon Jun 16 03:55:00 BST 2025

SECONDARY ACCESSION(S): PXD065040

REPOSITORIES: MassIVE