UGPase | UDP-glucose pyrophosphorylase (cytoplasm marker, dicots)

10 µg/well of total protein extracted from Arabidopsis thaliana leaf, using PEB extraction buffer (Agrisera AS08 300) and denatured with Invitrogen LDS sample buffer (4X) at 70°C/5 min.  Samples were separated on Invitrogen NuPage Bis-Tris 4-12% SDS-PAGE  and blotted for 1 h to Invitrogen PVDF (pore size of  0.45 µm), using: wet transfer. Blot was blocked with 5% milk in TBS-T for: 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 for 1h/RT with TBS-T Blocking. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG ALP conjugated, AS09 607 lot 2302 ) diluted to 1: 5 000 in TBS-T Blocking for 0,5h/RT with agitation. The blot was washed as above and developed with AS19 BCIP-NBT-PLUS lot 08225181 for 1 min. As soon as the desired band is detectable, briefly wash the membrane in generous amounts of deionized water. Transfer the membrane to fresh deionized water and incubate for 2 minutes with agitation before placing the membrane on Whatman paper to dry.

Courtesy Agrisera