Anti- Lhcb1 | LHCII type I chlorophyll a/b-binding protein

1 - MW marker
2 - 10 µg of Zea mays whole leaf extract
3 - 10 µg of Hordeum vulgare whole leaf extract
4 - 10 µg of thylakoid of Arabidopsis thaliana
5 - 10 µg of cyanobacterial whole cell extract (negative control)

10 µg/well of samples 1-5 were extracted and loaded in each well following denaturation 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 um), using wet transfer. Blot was blocked with 5% nonfat milk in TBS-T for: 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 500 for 4ªC/ON in 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 2503) diluted to 1: 1 000 in TBS-T Blocking for 0,5h/RT with agitation. The blot was washed as above and developed with AS19 BCIP-NBT-PLUS lot 03088241 for 3min. As soon as the desired band is detectable, briefly wash the membrane in generous amounts of deionized water. Image was captured after 2h.

Note: for more sensitive detection method the antibody can be used in higher dilution and lower protein load/well, which will also contribute to lower background signal.
Courtesy Agrisera