GDP-L-Galactose Phosphorylase

application example

9 ul of total soluble protein from Arabidopsis thaliana (1), Zea mays (2), Nicotiana tabacum (3), Citrus lemon (4), Spinacia oleracea (5), Brassica rapa var. komatsuna (6), Brassica oleracea var. italica (7) was separated on 12% SDS-PAGE and blotted 1.5h to PVDF at 1.5 mA/cm2 constant current. Blots were blocked immediately following transfer in TBS-0.3% Tween 20 + 5% dried milk overnight at room temperature (RT) with agitation. Blots were incubated in the primary antibody AS10 723 at a dilution of 1: 1500 for 4 h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly once with TBS-T+5%milk, followed by washing 3 times for 5 min in the same at RT with agitation. Blots were incubated in secondary antibodies (goat anti-rabbit IgG HRP conjugated from Biorad) diluted in TBS-T+5%milk to 1:25 000 for 1 h at RT with agitation. The blots were washed 2 x 5 mins with TBS-T+5% milk as above, then rinsed with TBS and followed by ECL detection (approx 5 minutes).

Courtesy Dr. Eugen Urzica, UCLA

GDP-L-Galactose Phosphorylase is a histidine triad (HIT) enzyme of the Smirnoff-Wheeler pathway of ascorbic acid synthesis in plants. Encoded by VTC2 gene. The enzyme catalyzes the conversion of GDP-L-galactose to L-galactose 1-phosphate in a reaction that consumes inorganic phosphate and produces GDP.