AS18 4235 Anti-AHB1 | Non-symbiotic hemoglobin 1 (other species)

20 or 10 µg/well of total protein extracted freshly from roots from 5 days seedlings (flooded for or without flooding – control)   with write exact buffer components 0.1 M Tris-HCl 7,5, 10 % glycerol, 1 mM EDTA, 2 mM DTT, 1 mM PMSF, 2 % PVPP and denatured with 62,5 mM Tris-HCl pH 6.8, 2 % SDS, 10 % glycerol, 20 mM DTT, 0,01 % bromophenol blue, at 98°C or 70°C for 5 min. Proteins were separated on 10 % SDS-PAGE  and blotted 2h (constant 90 V, 4°C) to PVDF/nitrocellulose (pore size of 0,2  um), using wet, semi-dry or dry transfer. Blot was blocked with 5 % milk or % BSA  for 1h/RT or 4°C/ON with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 for 1h/RT with agitation in TBS-T or ON/4°C 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 TBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:25 000 in  for 1h/RT with agitation. The blot was washed as above and developed for   min with Agrisera ECLBright or AgriseraECLSuperBright with   BioRad ChemiDoc XR S+. Exposure time was 40 seconds.

Courtesy of Dr. Paweł Staszek, SGGW, Warsaw, Poland

For detection in plant samples, plants need to be flooded as AHB1 levels in non-treated plants are very low.

The antibody is binding to human hemoglobin.

AHB1 (Non-sybmiotic hemoglobin) plays an important role in as nitric oxide scavenger and stress response mediator.

Agrisera Western Blot protocol and video tutorials

Protocols to work with plant and algal protein extracts

Agrisera Educational Poster Collection