CHLH (GUN5) | Magnesium-chelatase subunit ChlH, chloroplastic

Samples:
1 - 50 µg Hordeum vulgare whole leaf extract
2 - 50 µg Hordeum vulgare gun5 knock out whole leaf extract
3 - 30 µg Hordeum vulgare whole leaf extract
4 - 30 µg Hordeum vulgare gun5 knock out whole leaf extract
5 - 10 µg Hordeum vulgare whole leaf extract
6 - 10 µg Hordeum vulgare gun5 knock out whole leaf extract

Leaf material was homogenized in Laemmli sample buffer [20% (v/v) glycerol, 4% (w/v) SDS, 160 mM Tris–HCl pH 6.8, 10% (v/v) 2-mercaptoethanol] to a concentration of 0.1 mg µl−1 (fresh weight/Laemmli sample buffer). Samples were incubated at 65°C for 15 min and, after a centrifugation step (10 min at 16 000 g), the supernatant was incubated for 5 min at 95°C and loaded onto 10 % SDS-PAGE Acrylamide/Bis-acrylamide (29:1) and blotted for 1 h to PVDF (0.45 µm pore size), using semi-dry or dry transfer (Biorad). Blot was blocked with 5 % milk for: 4h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 5000 for ON/4°C with agitation. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 10 min in TBS-T at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated AS09 602, Agrisera) diluted to 1: 25 000 in for h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent: AS16 ECL-N-10 AgriseraBright (mid picogram). Exposure time was 2 minutes.

Courtesy of Dr. Luca Tadini, Università degli studi di Milano, Italy