Pet 23b bacterial expression vector

A C-term hPlk4 fragment (aa 510–970) was cloned into a pET-23b bacterial expression vector (EMD Millipore) containing a C-term 6×His tag. Recombinant protein was purified from Escherichia coli using Ni-NTA beads (QIAGEN) and used for immunization (Prosci). A STIL C-term peptide VGTFLDVKRLRQLPKLF (aa 1271–1287) was synthesized and conjugated to KLH for immunization. Rabbit immune sera were affinity-purified using standard procedures. Affinity-purified antibodies were directly conjugated to DyLight 550 and DyLight 650 fluorophores (Thermo Fisher Scientific) for use in immunofluorescence.
A synthetic phospho-peptide based on the human hSTIL sequence flanking serine 1108 [CDRSTVGL(pS)LISPN] or 1116 [CSPNNM(pS)FATKK] was synthesized, coupled to KLH, and injected into rabbits (Prosci). Polyclonal pS1108 and pS1116 antibodies were affinity-purified using the appropriate phosphopeptide coupled to a SulfoLink Coupling Resin (Thermo Fisher Scientific).

A C-terminal Plk4 fragment (aa 510–970) was cloned into a pET-23b bacterial expression vector (EMD Millipore) containing a C-terminal 6xHis tag. Recombinant protein was purified from Escherichia coli using Ni-NTA beads (QIAGEN) and used for immunization (ProSci). A STIL C-terminal peptide VGTFLDVKRLRQLPKLF (aa 1,271–1,287) was synthesized and conjugated to KLH for immunization. Rabbit immune sera were affinity purified using standard procedures. Affinity purified antibodies were directly conjugated to DyLight 550 and DyLight 650 fluorophores (Thermo Fisher Scientific) for use in immunofluorescence.