Antibody-mediated Identification of L5 Interactors

500 µg of wild type procyclic nuclear extract was used for each IC sample. An affinity-purified antibody against the L5 peptide ²⁹⁶VAAVIERIRDRAK³⁰⁸ (Bethyl Laboratories) was used. Dynabeads (Invitrogen) were cross-linked to anti-L5 antibody using dimethyl pimelimidate (DMP, Thermo Scientific) following the manufacturer’s instructions. As a negative control, no antibody was added to one reaction to assess the degree of non-specific interactions with the Dynabeads. Nuclear extracts were added to the Dynabeads coated with antibody and incubated overnight at 4°C. Supernatants (S) were collected and the beads were washed five times with phosphate-buffered saline (PBS). The Dynabeads were then resuspended in SDS-PAGE sample buffer and boiled for five minutes to dissociate interacting proteins. Finally, samples were subjected to denaturing gel electrophoresis. To detect the presence of P34 and P37 in association with L5, the proteins were transferred to a nitrocellulose membrane. The membrane was blocked with 10% non-fat milk and incubated with an anti-P34/P37 affinity purified, polyclonal antibody previously described [10] (link). A secondary goat anti-rabbit antibody conjugated to horseradish peroxidase (HRP) was used for detection in conjunction with the SuperSignal West Pico Chemiluminiscent Substrate (Thermo Scientific). These experiments were repeated three times and representative results are shown.
For the IC with recombinant proteins, 100 ng of recombinant L5 were incubated with 100 ng of recombinant P34 or P37 in the absence or presence of stoichiometric amounts of in vitro-transcribed 5S rRNA and immunoprecipitated with Dynabeads cross-linked to the L5 peptide antibody as indicated above. The experiments were repeated a minimum of three times with different preparations of recombinant proteins and synthetic 5S rRNAs.