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

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: Setting up the in vitro GNPT assay (A) Drying down of the [ 3 H]UDP-GlcNAc donor molecule mixed with cold UDP-GlcNAc and ATP using a Speed Vac Concentrator attached to a cold trap that is connected to a vacuum pump. (B) Sonicator with attached microprobe used to prepare whole cell lysate. (C) Equilibration of QAE Sephadex column. (D) Binding of the reaction mixture to the QAE Sephadex column and washing of the column. (E and F) First and second elutions with 2 mM Tris buffer containing 30 mM NaCl. (G) Detecting counts in the elutions using a liquid scintillation counter.

Article Snippet: QAE Sephadex A-25 , MilliporeSigma , Cat#GE17-0190-01.

Techniques: In Vitro, Binding Assay