GN | Gnom

Samples: M - Marker, A - Arabidopsis thaliana wild type, B - gnom R5 mutant, C gn:GNOM-GFP/gn:GNOM-3xHA The cell lysate was prepared from 10 A. thaliana seedlings (8-9d old). Seedlings were frozen in liq N2, then lysed with tissue lyzer, and 100 µl extraction buffer (100mM NaH2PO4 10mM Tris/HCl pH 8.0, 8M Harnstoff) was added. Total protein was denatured with 4x Laemmli buffer at 72ºC for 5min and separated on a 4-20% Mini Protean precast gel from BioRad and blotted for 4.5h to nitrocellulose using wet transfer. Blots were blocked with 5%NFDM for 1h at RT with agitation. The blot was incubated with primary antibody at a dilution of 1:1000 O/N at 4ºC in 1% NFDM in TBS-T. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once 15min and 3x 5min in TBS-T at RT with agitation. The blot was incubated using anti-rabbit IgG (HRP conjugated) from Agrisera (AS09 602) diluted 1:25 000 in 1% NFDM in TBS-T for 1h at RT with agitation. The blot was washed as above and developed for 5min with Agrisera ECLSuperBrigth Exposure time was 5 sec.
The cell lysate (GNOM-GFP/GNOM-3xHA) was prepared by grinding 1g seedlings(5-6 days old) in 1ml lysis buffer (50mM Tris-HCl pH7.5, 150mM Nacl+2mM EDTA+1% Tx 100 + Protease inhibitors). Total protein was denatured with 4x Laemmli buffer at 95C for 5min.

Courtesy of Dr. Antje Feller, University of Tübingen, Germany

Nitrocellulose membrane with pore size 0.45 µm is recommended as well as TBS-T as antibody incubation buffer. 

This antibody is also recognzing recombinant GN fused to GFP.