Anti mouse igg conjugated to horseradish peroxidase hrp

Recombinant proteins and fragments (4 μg) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Full-length PspA/Rx1 and PspA/Rx1 fragments were detected with the different monoclonal antibodies (10 μg/ml), MAbs 1b2.21, 8b2.19, Rx1MI003, Rx1MI005, Rx1MI006, and Rx1MI007, followed by incubation with anti-mouse IgG conjugated to horseradish peroxidase (HRP) (Sigma-Aldrich). Detection was performed using an enhanced chemiluminescence (ECL) kit (GE Healthcare).

For co-immunoprecipitation of potential mASAL interacting proteins the total cell lysate from R. solani cells were prepared as described before. One ml of cell lysate was incubated with 100 μg of purified recombinant mASAL at 4 °C overnight. Equilibrated Ni-NTA-agarose beads (Qiagen, Germany) were added to each lysate - protein mixture, further the reactions were allowed to rock slowly at 4 °C for 1 h. The beads were pelleted at 3000 × g for 10 min. The supernatant was discarded and the beads were washed twice with 500 μl of lysis buffer. Following this the beads were finally resuspended in 40 μl of 1X SDS-PAGE loading buffer and boiled for 10 min. After boiling the samples were centrifuged and the eluted proteins were separated by SDS-PAGE and immunoblotted onto a nitrocellulose membrane (Hybond-C, GE Healthcare). After blocking, the membranes were probed with primary antibodies against either ATPase or HSP70 or Actin (Pierce, USA). Following this each of the blots were incubated with anti-mouse IgG conjugated to horse radish peroxidase (HRP) (Sigma-Aldrich, USA) at 1:20,000 dilutions. Bands were detected by enhanced chemiluminescence (ECL) reagents (GE Healthcare, Germany).