Anti-PetL | Cytochrome b6-f complex subunit 6

8-16 µg/well of thylakoids extracted from Nicotiana tabacum. Exact buffer components were: 50 mM Hepes-KOH pH 7.5, 100 mM sorbitol, 10 mM MgCl2 and denatured with 0.1 M Tris/HCl pH 6.8, 0.24% glycerol, 8% SDS, 2% b-mercaptoethanol, 0.2 mg/ml Coomassie Blue G250 at 95 °C 10 min.  Samples were separated in the cold on 10 % SDS-tricine and blotted for overnight to PVDF, using wet transfer in the cold. Blot was blocked with 4 % milk for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1:  500 for ON/4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 10 min and 3 times in TBS-T at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602) diluted to 1: 25 000 1h/RT for 1h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent: AgriseraBright, AS16 ECL-N. Exposure time was 25 seconds.

The antibody needs to be used on a thylakoid fraction. No band is going to be obtained in Western blot on a total cell extract.