Anti-AGB1 | Guanine nucleotide-binding protein beta 1

Application example



10 µl of protein samples from Arabidopsis thaliana were separated on SDS-PAGE and blotted 1h to PVDF using tank transfer. Blots were blocked with Tris-buffered saline containing 0.05% Tween-20 (TBS-T) and 5% skimmed milk powder for 1h at room temperature (RT) with agitation. The blot was incubated in the primary antibodies indicated at a dilution of 1: 5000 overnight at 4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 5 times for 15 min in TBS-T with milk powder at RT with agitation. The blot was incubated in secondary antibody (Goat-Anti-Rabbit AP conjugate, Sigma) diluted 1:5000 for 2h at RT with agitation. The blot was washed as above with TBS-T without milk powder, equilibrated in AP buffer (100mM TRIS pH=9.5, 100mM NaCl, 50mM MgCl2) and then developed with AP substrate and imaged with a BioRad Chemi Doc Touch system. Exposure time was:  10 minutes.

Total protein from the indicated Arabidopsis thaliana lines was extracted with extraction buffer CE ( 250mM sucrose, 100mM HEPES-KOH pH 7.5, 5% glycerol, 1mM Na2MoO4 x 2H2O, 25mM NaF, 10mM EDTA, 1mM DTT, 0.5%Triton X-100, protease inhibitor cocktail). Protein concentration was measured with a Bradford assay and adjusted to 1mg/ml. Samples were denatured with SDS loading dye (50mM Tris-HCl pH6.8, 100mM DTT, 2%SDS, 10% glycerol, 0.025% bromophenol blue) at 70°C for 2-5 min.

Courtesy of Dr. Elena Petusching, Georg-August-University Goettingen, Germany

AGB1 belongs to a family of proteins involved in signal transduction. They are acting like molecular switches when transmitting signals from the outside to the inside of cells.