Anti-L22 | 50S ribosomal protein L22 ( chloroplastic)

Samples:

20 µg/well of total protein extracted freshly from 08-day-old Arabidopsis thaliana seedlings using 1% SDS and 8M urea. Proteins eluted in 8M urea (w/o SDS) were denatured with 2x Laemmli buffer (LB) at 75 °C for 7 min. Samples were separated using SDS-PAGE (20% gel) and blotted for 1.5 h to nitrocellulose (pore size of 0.45 µm), using wet transfer in the cold. Blot was blocked with 5% milk for ON/4ºC with agitation. Blot was incubated in the primary antibody at a dilution of 1:1000 for 7 h/4°C with agitation (5% Blotto in TBS-T). The antibody solution was decanted, and the blot was rinsed briefly twice and washed thrice for 15 min each in TBS-T at RT. Blot was incubated in anti-rabbit IgG HRP-conjugated secondary antibody diluted to 1:15 000 for 1 h/RT with agitation. The blot was washed thrice for 15 min each in TBS-T at RT and developed with the following chemiluminescent detection reagent: AS16 ECL-N-10 AgriseraBright. The exposure time was 30 seconds.

The middle band is L22 protein. Band pattern can vary depending upon analyzed material and applied protocol. 

Courtesy of Project Associate. Kavyashree A and Dr. Naresh Loudya, Indian Institute of Science, Bengaluru, India

Samples:
1 - 15 ug of Arabidopsis thaliana whole seedling extract (Control)
2 - 15 ug of Arabidopsis thaliana whole seedling treated with drug-1 for 16hr
3 - 15 ug of Arabidopsis thaliana whole seedling treated with drug-2 for 16hr
4 - 15 ug of Arabidopsis thaliana whole seedling and enrich to chloroplast

15 µg/well of total protein extracted freshly from Arabidopsis thaliana 8-day-old seedlings. Exact buffer components were: 8M Urea buffer components at 4 °C. Samples were separated in the RT on 10 % SDS-PAGE and blotted for 2 h to PVDF (pore size of 0.2 µm), using: we transfer in the cold. Blot was blocked with 10 % milk for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 3000 for 1h/RT with agitation in PBS with agitation. 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 PBS at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1: 5000 in for 1 h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent. Exposure time was 1 minute.

L22 protein band is a middle band, clearly visible in enriched chloroplast fraction. Longer exposure time will visualize bands in all samples. 

Courtesy of Dr. Kuo-En Chen Department of Biology, Washington State University in St. Louis, USA