Journal: STAR Protocols
Article Title: Protocol to measure endogenous GlcNAc-1-phosphotransferase activity in SK-MEL-30 cells
doi: 10.1016/j.xpro.2025.103935
Figure Lengend Snippet: GlcNAc-1-phosphotransferase enzyme activity assay (A) Schematic diagram of the in vitro GNPT enzyme assay using with α-MM acceptor. The activity of endogenous GNPT in SK-MEL-30 whole cell lysates is measured to quantify the amount of α-MM-P-[ 3 H]GlcNAC reaction product that is formed in 1 hr. (B) The unreacted [ 3 H]UDP-GlcNAc donor molecule with two phosphates remains bound to the QAE-Sephadex column, while the α-MM-P-[ 3 H]GlcNAc reaction product with one phosphate elutes with 30 mM NaCl. ∗Note that there is invariably spontaneous breakdown of a very small amount of the [ 3 H]UDP-GlcNAc to [ 3 H]GlcNAc-1-P and UMP. The [ 3 H]GlcNAc-1-P will also bind to the column and elute with 30 mM NaCl to yield the background counts.
Article Snippet: QAE Sephadex A-25 , MilliporeSigma , Cat#GE17-0190-01.
Techniques: Enzyme Activity Assay, In Vitro, Enzymatic Assay, Activity Assay