Anti-Raptor 1/2 | Regulatory-associated protein of TOR 1/2

Samples:
1-3: 10 µg protein from Col-0 ·
4-9: 10 µg protein from misc mutants 
10-12: 10 µg protein of RAPTOR1b knockout mutant

5-15 µg/well of total protein was extracted freshly from Arabidopsis thaliana root tips. Exact buffer components were: TBS, PhosStop, and Protease inhibitor cocktail and denatured with Laemmli buffer with 10% BME at 95°C/5 min. Samples were separated at room temperature on 8 % SDS-PAGE and blotted for 1.5 hrs to nitrocellulose (pore size of .45 um), using: wet transfer in the cold. Blot was blocked with 5 % milk for: 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1:1000 ON/4°C with agitation. The antibody solution was decanted, and the blot was rinsed briefly three times, 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) diluted to 1: 5000 in for 2 h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent: Pierce™ ECL Plus Western Blotting Substrate. Exposure time was by automatic detection.

Notes:
It has not been ruled out if a band in Raptor 1b mutant is Raptor 1a protein. Double Raptor 1a/b mutant may be lethal. 

PhosSTOP is a proprietary blend of phosphatase inhibitors, formulated as a ready-to-use, quick-dissolving, water-soluble tablet. Comprehensive protection: Inhibit acid and alkaline phosphatases, as well as serine/threonine (PP1, PP2A, and PP2B) and tyrosine protein phosphatases.

Courtesy of Dr. Brandon Reagan, Michigan State University, USA