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 

Samples: 

1 - 10 ug of 4 days old of wild-type Arabidopsis thaliana seedlings extract

2 - 5 ug of 4 days old of wild-type Arabidopsis thaliana seedlings extract

3- 10 ug of 4 days old of gun5-1 mutant seedlings extract

Mark: MW markers

Target MW: 153.57 kDa

5-10 µg/well of total protein extracted from fresh 4 days old of Arabidopsis thaliana whole seedling.  Exact buffer components were: (50 mM Tris–HCl pH 7.5, 10% glycerol, 150 mM NaCl, 10 mM MgCl2, 5 mM EDTA, 5 mM DTT, 0.5% (v/v) Triton X-100, and 1 × protease inhibitors) and denatured with 4X SDS sample loading buffer (200 mM Tris-Cl (pH 6.8). 8% SDS (sodium dodecyl sulfate). 0.4% Bromophenol blue. 40% glycerol) at 95°C 10 min.  Samples were separated in the RT on 10 % SDS-PAGE and blotted for 0.5 h to PVDF (pore size of 0.45 um), using:  semi-dry at room temperature. Blot was blocked with 5 % milk for: 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1:  2 000 at 4°C with agitation overnight. 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 horse radish peroxidase conjugated, AS09 602 Agrisera) diluted to 1: 50 000 for 2 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. Duorong Xu, LMU München, Germany