Hexokinase Activity Assay in Mouse Retina

The hexokinase assay was carried out as described (49 (link)). Mouse retinas were lysed in 50 mM potassium phosphate, 2 mM DTT, 2 mM EDTA, and 20 mM NaF. The assay was carried out in the presence of mouse retinal lysate containing an enzyme buffer mixture [100 mM Tris–HCl (pH 8.0), 10 mM MgCl₂, 0.5 mM EDTA, 10 mM ATP, and 0.2 mM NADP], 2 mM D-glucose, and 1 U glucose 6-phosphate dehydrogenase. The hexokinase activity was measured at 340 nm spectrophotometrically by monitoring the generation of NADPH.

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Rajala A., Bhat M.A., Teel K., Gopinadhan Nair G.K., Purcell L, & Rajala R.V. (2023). The function of lactate dehydrogenase A in retinal neurons: implications to retinal degenerative diseases. PNAS Nexus, 2(3), pgad038.

Publication 2023

Assay Buffer Edta Enzyme Glucose Glucose 6 phosphate dehydrogenase Hexokinase Mgcl2 Mouse Nadph Potassium phosphate Retinal Tris

Corresponding Organization :

Other organizations : Dean McGee Eye Institute, University of Oklahoma Health Sciences Center

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Variable analysis

independent variables

Presence of mouse retinal lysate

dependent variables

Hexokinase activity measured at 340 nm spectrophotometrically

control variables

Enzyme buffer mixture [100 mM Tris–HCl (pH 8.0), 10 mM MgCl2, 0.5 mM EDTA, 10 mM ATP, and 0.2 mM NADP]
2 mM D-glucose
1 U glucose 6-phosphate dehydrogenase

controls

Positive control: Mouse retinal lysate containing the enzyme buffer mixture, D-glucose, and glucose 6-phosphate dehydrogenase
Negative control: Not explicitly mentioned

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