Anti-Halo Tag, derived from DhaA of Rhodococcus rhodochrous

Samples:
1. Ladder (PageRuler Prestained, 10 à 180 kDa - Thermo)
2. Non induced bacteria
3. Induced bacteria at 37°C for 3h
4. Aliquot lysate (after sonication) supernatant
5. Flowthrough after incubation with beads
6. Elution 1 with acidic elution buffer
7. Elution 2 with acidic elution buffer
8. Elution of beads with 2x SDS-sample buffer

Recombinant protein was purified from 25 ml E. coli Rossetta bacteria induced at 37°C during 3 h. After sonication of the culture (lysate), Halo-Trap Agarose beads (chromotek) were added to the supernatant and incubated during 1 h at 4°C. After 3 wash, beads were eluted with an acidic buffer (x2) and with 50 μl laemmli 2X). Samples were then separated on 12 % SDS-PAGE and blotted for 1 h to nitrocellulose (pore size of 0.45 um), using wet transfer. Blot was blocked with 5% milk for 2 h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1000 in 5% milk TBS-T ON/4°C with agitation. The antibody solution was decanted, and the blot was washed 4 times for 5 min in TBS-T at RT with agitation. Blot was incubated with secondary antibody (AS09 602, Rabbit anti-mouse IgG, HRP conjugated) diluted to 1: 20000 in 5% milk for 1h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent AS16 ECL-S-10, AgriseraBright. Exposure time was 30 seconds.

Courtesy of Paul Schouvelier, IBPS, Equipe Biologie de Semences, France