Anti-Api g 1 | Major allergen Api g 1, isoallergen 2

Samples:
1 – 12 µg celery (Apium graveolens) leaves, proteins extracted with 10% TCA/Acetone
2 – 12 µg (Apium graveolens) stems, proteins extracted with 10% TCA/Acetone
3- 12 µg (Apium graveolens) leaves, proteins extracted with 50mM Tris-HCl, pH7.4; 150 mM NaCl, protease inhibitors, followed by protamine sulfate depletion (0.1% for RuBisCO removal) and precipitated with 10% TCA/Acetone
4- 12 µg (Apium graveolens) leaves, proteins extracted with 50mM Tris-HCl, pH7.4; 150 mM NaCl, protease inhibitors
MW marker (kDa): PageRuler™ Prestained Protein Ladder (Thermo Fisher Scientific)

12 µg/well of total protein extracted freshly from celery (Apium graveolens) with 10% TCA/ Actetone (samples 1 and 2) and with 50mM Tris-HCl, pH 7.4; 150 mM NaCl, protease inhibitors, followed by protamine sulfate depletion (0.1% for RuBisCO removal) and precipitated with 10% TCA/Acetone (samples 3 and 4) and denatured in Laemmli buffer at 70°C for 5 min. The samples were separated on 10% SDS-PAGE and blotted 1 h to nitrocellulose using wet transfer. Blot was blocked with 2 % milk TBS-T at 4°C/ON without agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 for 1h/RT with agitation in TBS-T 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 horseradish peroxidase conjugated, AS09 602) diluted to 1:25 000 in TBS-T for 1h/RT with agitation. The blot was washed as above and developed for 5 min with Agrisera ECL SuperBright. Exposure time was 60 seconds.

Courtesy of Yordan Muhovsky, Walloon Agricultural Research Centre CRA-W · Department of Life Sciences, Belgium