anti-HSP90 | Heat shock protein 90 (cytoplasmic, monocot)

15-25 μg/well for rice and 5-10 μg/well for banana of total protein was extracted freshly from leaf sample (rice leaf taken from field; banana leaf taken from soil pot). Exact buffer components were 50mM Tris-Cl (ph 7.5), 150 mM NaCl, 10 mM MgCl2, 1 mM EDTA (pH 8.0), 10% Glycerol 0.1% Nonidet P40, 14 mM 2-betamercaptoethanol, 1X protein inhibitor cocktail, 40 μM MG132, and denatured at 95 °C for 10 min. Samples were separated in the RT at 10% SDS-PAGE and blotted for 3 h at 4°C at 300mA to nitrocellulose (pore size of 0.45 um), using wet transfer in the cold. Blot was blocked with 5% BSA for 2h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 5000 with agitation in PBS-T for ON/4°C. The antibody solution was decanted and the blot was washed once for 15 min and 3 times for 5 min in PBS-T at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1: 10 000 in for 2h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent. Exposure time was 2 minutes.

Courtesy of Dr. Jogindra Naik, Plant Metabolic Engineering Group National Institute of Plant Genome Research, New Delhi, India