His spintrap kit

His-MBP-CERKL fusion proteins were produced in E. coli and purified with the His SpinTrap kit (GE Healthcare). Messenger RNA was purified from total RNA from COS-7 cells and from human retina using the Oligotex mRNA kit (Qiagen) and biotinylated using the RNA 3′ biotinylation kit (Thermo Fisher Scientific). EMSA was conducted using LightShift Chemiluminescent RNA EMSA kit (Thermo Fisher Scientific) according to the manufacturer's protocol. BSA was used as a negative control. The protein-probes complexes were resolved in a 6% polyacrylamide native gel and transferred to a Hybond-N⁺ Nylon membrane (GE Healthcare). Migration of biotin-labeled probes was detected on an ImageQuant™ LAS 4000 biomolecular imager (GE Healthcare) using streptavidin-horseradish peroxidase conjugates and chemiluminescent substrates for biotin recognition.

The proteins were expressed in the Escherichia coli strain Top 10 (Thermo Fisher Scientific, Waltham, MA, USA) using the pBAD/HisB or in the E. coli strain SURE (Agilent, Santa Clara, USA) using the pQE31 expression plasmid. E. coli cells were grown at 37 °C in lysogeny broth (LB) medium and protein expression was induced with 0.02% L-Arabinose or 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG).
The FPs were purified by Ni-NTA affinity chromatography (His SpinTrap Kit, GE Healthcare, Little Chalfont, BKM, GB) according to the manufacturer’s instructions with a 30 min binding step. The purified proteins were concentrated by ultrafiltration using Vivaspin 500 columns (Sartorius, Göttingen, DE) and taken up in 100 mM Tris-HCl, 150 mM NaCl pH 7.5.

The GST fusion proteins were expressed in bacteria and purified using a glutathione affinity matrix as described previously65 (link)74 (link). GST fusion proteins immobilized on the glutathione resin were either used immediately or stored at 4 °C for no longer than 3 days. Each batch of fusion proteins used in experiments was first analyzed by Coomassie Brilliant Blue R250 staining following SDS-PAGE. GST fusion proteins tethered to the glutathione resin were incubated with total cell lysates in 500 μl of binding buffer containing 20 mM Tris-HCl, pH 7.5, 1% NP-40, 140 mM NaCl, 1 mM MgCl₂ and 0.5% bovine serum albumin at 4 °C for 4–6 h. The resin was washed four times with 0.5 ml of binding buffer and the retained proteins were solubilized in SDS-gel loading buffer and separated by SDS-PAGE. Proteins bound to GST fusion proteins were detected by immunoblotting.
To determine if GGAs could directly interact with α_2B-AR, GGA1 and GGA2 were tagged with the epitope His at their N-termini in the pET-28 vector and purified by using His SpinTrap kit (GE Healthcare) as described previously77 (link). About 1 μg of purified His-GGAs was incubated with GST-ICL3 fusion proteins in 600 μl of binding buffer at 4 °C for 4 h and the retained His-GGAs were measured by immunoblotting using ant-His antibodies.