NADP-ME | NADP-malic enzyme, chloroplastic (dicots/monocots)

Arabidopsis thaliana, Beta vulgaris subsp. Vulgaris, Citrus clementina,Cynara cardunculus var. ScolymusKalanchoe fedtschenkoi, Elaeis guineensis, Elaeis guineensis,Lactuca sativa, Flaveria robusta, Flaveria pringlei, Flaveria floridana, Flaveria brownii, Flaveria palmeri, Flaveria linearis, Glycine max,Glycine soja,Helianthus annuus, Hibiscus syriacus, Lupinus angustifolius, Musa acuminata subsp. Malaccensis, Vigna angularis, Phtheirospermum japonicum, Sesamum indicum, Spatholobus suberectus,Vigna unguiculata, Vigna radiata var. Radiata, Vigna angularis

Monocots: Hordeum vulgare, Oryza sativa, Setaria viridis, Zea mays

Samples:
Flaveria bidentis whole leaf extract - 1
Zea mays whole leaf extract - 2
Neurachne muelleri whole leaf extract - 3

5 ug/well of total protein extracted from leaves of species listed above, were extracted in 50 mM HEPES, pH 7.1, 1% (w/v) polyvinylpolypyrrolidone, 1 mM EDTA, 10 mM DTT, 5 mM MgCl2, 1 mM PMSF and denatured with 0.5 M Tris-HCl pH 6.8, 10% (w/v) SDS, 10% (v/v) glycerol, 0.001% (w/v) bromophenol blue at 90°C for 5 min. Samples were separated on 10% SDS-PAG and blotted for 1 h to nitrocellulose (pore size of 0.45 um), using semi-dry transfer. Blot was blocked with 5% (w/v) skim milk powder in TBS-T for 1 h at RT with agitation. Blot was incubated in the primary antibody (rabbit anti NADP-ME, chloroplastic, affinity purified serum, AS21 4580 lot 2206) at a dilution of 1: 5 000 in TBS-T ON at 4°C with agitation. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed 3 times for 5 min each time in TBS-T at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1: 25 000 in for 1 h at room RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent: Agrisera AgriseraBright. Exposure time was 30 seconds or 5 minutes.

Courtesy of Prof Martha Ludwig, University of Western Australia, Australia

Protein was extracted from leaves of Arabidopsis thaliana, Oryza sativa and Neurachne alopecuroidea in protein extraction buffer (50 mM HEPES, pH 7.1, 1% (w/v) polyvinylpolypyrrolidone, 1 mM EDTA, 10 mM DTT, 5 mM MgCl2, 1 mM PMSF) and flash frozen at -80°C. Aliquots of protein extract were thawed and denatured in SDS loading buffer at 90°C for 5 minutes. 15 ug of total protein was loaded per well and separated on  10 % SDS-PAGE  and blotted for 1.5 h to nitrocellulose (pore size of  0.45 um), using semi-dry transfer. Blot was blocked with 5 % milk in TBS-T for 1h/RT or with agitation. Blot was incubated in primary at a dilution of 1:  5 000 in TBS-T ON/4°C with agitation. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in  matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1: 25 000 in TBS-T for 1h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent: Agrisera AgriseraBright. Exposure time was 1 minute.
Although C3 plants make several homologues of NADP-ME, they do not express high levels of the homologue that is plentiful in the C4 species. This explains the weak labelling even when a relatively high amount of leaf protein extract is on the blots.

Courtesy of Prof Martha Ludwig, University of Western Australia, Australia