Nickel nta resin

Soluble PCDH1 variants cloned into pcDNA3.1 (see above) were expressed in 293 F cells in shaker flasks by transient transfection with linear polyethyleneimine (Polysciences) and purified by nickel-chelation chromatography. Cell cultures were incubated at 37 °C and 8% CO₂ for six days post-transfection. Cell supernatants were clarified and stirred overnight at 4 °C with proteinase inhibitor (Sigma-Aldrich) and nickel-NTA resin (Qiagen) at 0.3 mL packed resin per 50 mL cell supernatant. Nickel-NTA beads were then collected, washed with phosphate buffer saline (PBS) containing 50 mM imidazole, and eluted with PBS containing 250 mM imidazole. The eluted protein was buffer-exchanged with PBS, concentrated, and stored in aliquots at −80 °C. The purity of the secreted PCDH1 variants was determined by size-exclusion chromatography (SEC) and/or either SDS-PAGE or Native-PAGE gels, stained with Bio-Safe™ Coomassie G-250 Stain (Bio-Rad) and imaged on a LI-COR OdysseyⓇ Fc Imager (LI-COR Biosciences, LICOR Image Studio software, V.1.0.19). For analytical SEC, a Superdex S200 (10/300) column was equilibrated in PBS and calibrated with Gel Filtration Standard (Bio-Rad) composed of thyroglobulin (MW 670 kDa), bovine γ-globulin (MW 158 kDa), chicken ovalbumin (MW 44 kDa), horse myoglobin (MW 17 kDa), and vitamin B12 (MW 1.35 kDa).

Anti-DNAJB3 antibody was purchased from Proteintech (Proteintech Group, Inc., Chicago, IL). Anti-AKT antibody was purchased from Cell Signaling (Cell Signaling Technology, Inc., Danvers, MA). Horse radish conjugated anti-GST antibody was purchased from Abcam (Abcam, Cambridge, UK). Insulin, wortmannin, ly294002, protease inhibitor cocktail, imidazole and reduced glutathione were purchased from Sigma (Sigma-Aldrich, St. Louis, MO). C2C12, HepG2 and 3T3-L1 were purchased from ATCC (ATCC, Manassas, VA). E. coli strains 10β and BL21 (DE3) were purchased from New England Biolabs (New England Biolabs, Ipswich, MA, USA). Glutathione resin was purchased from Pierce (Pierce, Rockford, IL, USA). Nickel-NTA resin, DNaseI and RNase A were purchased from Qiagen (Qiagen, Inc., Valencia, CA). Fluorescently labeled D-glucose analog (2-NBDG) was purchased from Cayman (Cayman, Ann Arbor, MI). Scrambled and specific siRNA were purchased from Dharmacon (Dharmacon Inc., Lafayette, CO). Lipofectamine 3000 and lipofectamine RNAiMAX were purchased from Invitrogen (Invitrogen, Carlsbad, CA). PureLinkTM RNA Minikit and High-Capacity cDNA Reverse Transcription Kit were purchased from Invitrogen (Invitrogen, Carlsbad, CA).

E. coli expression clones encoding recombinant human zinc finger genes ZNF346, ZNF638, ZNF700 and ZNF768 were obtained from imaGenes GmbH (Berlin, Germany) and were verified by sequencing (SourceBioscience Sequencing, Dublin, Ireland). ZNF proteins were overexpressed in E. coli and purified using nickel-NTA affinity chromatography. Briefly, auto-induction media (Tryptone 1% (w/v), Yeast 0.5%, 1mM MgSO₄, 1x 5052, 1x NPS, 100 μg ml^-1 carbenicillin and 50 μg ml^-1 kanamycin) were inoculated with E. coli expression clones and incubated in a shaker incubator overnight (37°C, 200rpm). Bacteria were harvested after 24 hours by centrifugation (12,000g, 30 min), pellets were snap frozen in liquid nitrogen and stored at -20°C. Proteins were purified under denaturing conditions with 6M guanidine-HCl using nickel-NTA resin (Qiagen) and were size-verified using SDS-PAGE and Instant Blue staining. Protein concentrations were determined using a Nanodrop spectrophotometer (ND-1000, Thermo Scientific) at an absorbance of 280nm. Purified proteins were stored at -20°C.

For anti-Gβ experiments, purification of His tag-β2-AR-G protein and control (−) sensors from HEK293T cells followed the previously published protocol^{16 (link)}. Frozen membranes were thawed quickly to room temperature and briefly re-homogenized with a rotary pestle. 5% cholate buffer (5% sodium cholate in 50 mM HEPES, 3 mM MgCl₂, 50 mM NaCl with 1 μg mL⁻¹ aprotinin, 1 μg mL⁻¹ leupeptin, 10 μg mL⁻¹ phenylmethanesulfonyl fluoride, and 5.5 mM β-mercaptoethanol, pH 8.0) was added to a final concentration of 1% cholate. This mixture was incubated on ice for 45 min and separated by ultracentrifugation at ~105,000 g for 40 min at 4 °C. The supernatant was harvested and diluted drop wise with four volumes of Buffer C to one volume of supernatant, and was pipetted gently to mix. The diluted supernatant was added to nickel-NTA resin (Qiagen) and incubated 30 min at 4 °C with rotation. The resin was washed 3x with 500 μL Buffer C + 5 mM imidazole. The final wash was removed and the resin was brought to room temperature and eluted for 3–5 min with 150 μL of elution buffer (Buffer C + 200 mM imidazole). The eluted resin was spun down, the supernatant harvested, and measured for mCerulean and mCitrine fluorescence in a fluorometer (as described above). Samples were stored in SDS laemmli sample buffer at −80 °C.

Soluble PCDH1 variants cloned into pcDNA3.1 (see above) were expressed in 293 F cells in shaker flasks by transient transfection with linear polyethyleneimine (Polysciences) and purified by nickel-chelation chromatography. Cell cultures were incubated at 37 °C and 8% CO₂ for six days post-transfection. Cell supernatants were clarified and stirred overnight at 4 °C with proteinase inhibitor (Sigma-Aldrich) and nickel-NTA resin (Qiagen) at 0.3 mL packed resin per 50 mL cell supernatant. Nickel-NTA beads were then collected, washed with phosphate buffer saline (PBS) containing 50 mM imidazole, and eluted with PBS containing 250 mM imidazole. The eluted protein was buffer-exchanged with PBS, concentrated, and stored in aliquots at −80 °C. The purity of the secreted PCDH1 variants was determined by size-exclusion chromatography (SEC) and/or either SDS-PAGE or Native-PAGE gels, stained with Bio-Safe™ Coomassie G-250 Stain (Bio-Rad) and imaged on a LI-COR OdysseyⓇ Fc Imager (LI-COR Biosciences, LICOR Image Studio software, V.1.0.19). For analytical SEC, a Superdex S200 (10/300) column was equilibrated in PBS and calibrated with Gel Filtration Standard (Bio-Rad) composed of thyroglobulin (MW 670 kDa), bovine γ-globulin (MW 158 kDa), chicken ovalbumin (MW 44 kDa), horse myoglobin (MW 17 kDa), and vitamin B12 (MW 1.35 kDa).

pET28b+ derivatives encoding Bph-H6 variants were transformed into E. coli BL21 (DE3) cells and grown in LB/kanamycin. Cultures were grown at 37 ^oC on an Innova 2300 platform floor shaker (New Brunswick Scientific) at 150 rpm to an OD₆₀₀ of 0.4–0.6, at which point 1.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to induce protein expression for 3 h. Cells were harvested by centrifugation (6371 × g for 10 min) and resuspended in lysis buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.01% Triton X-100, 20 mM imidazole, 100 μg/mL lysozyme, 1 mM PMSF, 10 U/mL DNase). Cells were broken by sonication in an ice bath using a QSonica Q500 sonicator (1.6 mm tip, 40% amplitude, 10 s on/20 s off for a total of 2 min pulse time), and cell debris was pelleted by centrifugation (30,000 × g for 15 min). Supernatants were transferred to tubes containing pre-washed nickel NTA resin (Qiagen) and incubated with rotation at 4 ^oC. The resin was washed 3 × with wash buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.01% Triton X-100, 20 mM imidazole), and proteins were eluted (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 250 mM imidazole). Fractions were analyzed for purity using Tris–glycine SDS–PAGE and dialyzed against 2 L buffer containing Tris–HCl pH 7.5, 150 mM NaCl. Proteins were stored at −20 ^oC in dialysis buffer plus 50% glycerol (v/v).