Nickel sepharose column

The region encoding amino acid residues 206–282 of the FcγRIIa cytoplasmic domain was PCR-amplified from pCMV FcγRIIa IRES neo and cloned into the bacterial expression vector pQE30-GB1 (Qiagen) in front of a histidine tag. The resulting construct was transduced into E. coli BL21 cells, induced with IPTG and purified from bacterial lysates using a nickel-Sepharose column (GE Healthcare Life Sciences). For kinase assays, recombinant FcγRIIa cytoplasmic domain proteins (1 mM) were incubated with Src, Lyn or Fyn in kinase assay buffer (250 μM ATP, 1 mM EGTA, 10 mM MgCl₂, 0.01% Brij 35, and 250 μM Na₃VO₄) for 60 min at 30°C and then boiled in the presence of an equal volume of 2× SDS-PAGE sample reducing buffer. The resulting products were separated on a 12% SDS−polyacrylamide gel and stained with Coomassie blue. Target bands were cut out, digested with trypsin, and subjected to mass spectrometric analysis following a previously described protocol [23 (link)].

The recombinant protein PvGAMA-Ecto domain comprising amino acid 22–771 was expressed and purified as previously described [13 (link)]. Briefly, the PVX_088910 fragments encoding PvGAMA-ectodomain and a hexahistidine (His) tag at the C-terminus, were amplified using sense primers with XhoI sites and antisense primers with BamHI restriction sites. The amplified fragments were then restricted and ligated into the WGCF expression vector pEU-E01-His-TEV-MCS (CellFree Sciences, Matsuyama, Japan). The cloned inserts were sequenced using an ABI 3700 Genetic Analyzer (Genotech, Daejeon, Korea). The recombinant proteins with His tags were expressed using a WGCF system (CellFree Sciences) and purified using a Nickel-Sepharose column (GE Healthcare Life Sciences, Uppsala, Sweden).

Erythrocytes were hypotonically lysed and centrifuged (16,000× g, 20 min, 4 °C). For depletion of hemoglobin, the supernatant was passed over a Nickel Sepharose column (GE Healthcare, Chicago, IL, USA), and eluted proteins were concentrated by precipitation with trichloroacetic acid. Samples were resuspended in 0.1% SDS and protein concentration determined via NanoDrop A280. Protein samples were separated by SDS PAGE and transferred to nitrocellulose (GE Healthcare). Membranes were blocked with 5% low-fat dry milk in TBS (150 mM NaCl, 10 mM Tris pH = 7.4), incubated overnight at 4 °C with anti-PANK2 antibody (sc-82288, Santa Cruz Biotechnology, Dallas, TX, USA), and a peroxidase-conjugated goat anti-rabbit IgG, antibody (Jackson ImmunoResearch, West Grove, PA, USA) at room temperature. The chemiluminescence reaction (Pierce ECL Western Blotting Substrate; ThermoFisherScientific) was quantified in a ChemiDoc system (Bio-Rad Laboratories, Hercules, CA, USA). Carbonic anhydrase (CA, rabbit monoclonal anti-carbonic anhydrase 1/CA1 antibody; ab108367, Abcam, Cambridge, UK) was used as a loading control. Optical intensity of the bands was quantified using Image Lab software (Version 6.1, BioRad).