Ni nta resin

Harvested cells were resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 5 % glycerol) with a protease inhibitor cocktail (Roche), and lysed by 4 cycles of freezing and thawing. Cell lysates were subsequently cleared by centrifugation at 39,000 × g for 1.5 h, and bound to Ni-NTA resin (Qiagen) pre-equilibrated with lysis buffer for 2 h. Ni-NTA resin was washed with wash buffer (lysis buffer supplemented with 20 mM imidazole) and elution buffer (lysis buffer supplemented with 100 mM imidazole). Eluted fractions were pooled and cut with TEV protease overnight in dialysis buffer (50 mM Tris-HCl pH8.0, 250 mM NaCl, 5% glycerol, 1 mM DTT). Contaminants and uncut His-tagged protein were removed by cycling over Ni-NTA resin 5 times, and further purified by running a 50 mM to 1000 mM NaCl gradient over a HiTrapQ 5 mL column (GE Healthcare). Typical yields were ~50 μg of protein per liter culture of insect cells.

Cells were pelleted by centrifugation at 4°C and resuspended in lysis buffer consisting of 50 mM Na₂HPO₄, 300 mM NaCl, 10 mM imidazole, 1 mM PMSF protease inhibitors, and 1 μl of 1 mg/ml DNase I for each ml of cell suspension, at pH 8. Resuspended samples were kept on ice and lysed by sonication. After sonication, the samples were centrifuged at 12,000 ×g for 1 h at 4°C, the supernatant was decanted, filtered through a 0.2 μm filter, and the pellet discarded. Lysate was mixed with Ni-NTA resin (Qiagen) (Ni-NTA resin volume was in the range of 4–8 ml of slurry mixture depending on the expected protein amount in the cell lysate) which had been previously equilibrated with 20 bed volumes of lysis buffer (50 mM Na₂HPO₄, 300 mM NaCl, 10 mM Imidazole, pH 8). The lysate-resin mixture was incubated at 4°C for 2 h (batch purification) and subsequently was applied by gravity flow to a polypropylene column. The column was washed with 20 bed volumes of washing buffer (50 mM Na₂HPO₄, 300 mM NaCl, 20 mM Imidazole, pH 8) followed by elution with three bed volumes of elution buffer (50 mM Na₂HPO₄, 300 mM NaCl, 300 mM Imidazole, pH 8). The eluted protein was buffer-exchanged against 20 mM Tris-Cl and 20 mM NaCl, pH 7.5 using 10 kDa MWCO protein concentrator tubes (Amicon) and was subjected to ion-exchange chromatography described below.

Purification of BLC23O was achieved
in two steps: an affinity chromatography
step and a size exclusion chromatography step. Ni-NTA resin (Qiagen,
Hilden, Germany) was used for the affinity purification step, in which
BLC23O with a 6xHis-tag binds to the Ni-NTA resin under a low concentration
of imidazole (10 mM) solution and was eluted with a high concentration
of imidazole (300 mM) solution. The enzyme was then further purified
through size-exclusion chromatography on an FPLC system (UPC-900 ÄKTA,
Amersham Pharmacia Biotech, Amersham, United Kingdom) using a HiLoad
16/60 Superdex 200 prep grade column (Cytiva, Malborough, MA, United
States). The buffer used for FPLC was composed of 10 mM Tris-HCl and
150 mM NaCl. Following purification, SDS-PAGE was performed to identify
the fractions that contain the target enzyme, as well as to determine
purity (See Figure S1). The protein sample
was then buffer exchanged (10 kDa MWCO) and concentrated to prepare
a protein stock in 10 mM Tris-HCl and at pH 7.4, which was stored
at 4 °C. Protein concentration was determined using the Bradford
protein assay^{25 (link)} with bovine serum albumin
(BSA) as the standard (See Figure S2).

Full-length GIPC-2 protein with six histidine residues (plasmid pET21b-MYC::GIPC-2) was expressed in and purified from E. coli BL21 (DE3) using Ni-NTA resin (Qiagen 30210), then used to raise rabbit polyclonal antibody. The anti-GIPC-2 antibody recognized both GIPC-1 and GIPC-2 in a western blot analysis using recombinant proteins. The antibody was further purified as described previously [52 (link)]. A C-terminal fragment of SPE-15a protein (amino acids 838–1219) fused with 6 histidine residues was expressed in and purified from E. coli BL21 (DE3) using Ni-NTA resin (Qiagen 30210), then used to raise mouse monoclonal antibody.

The Mre11-Rad50-Xrs2 proteins were expressed as a complex in Spodoptera frugiperda 9 (Sf9) cells. The Mre11 construct contained a His-tag at the C-terminus of Mre11, and a FLAG-tag at the C-terminus of Xrs2. The proteins were then purified using affinity chromatography using NiNTA resin (Qiagen) followed by anti-FLAG M2 affinity resin (Sigma)^{117 (link)}. Non-phosphorylated Sae2 (MBP-tagged at the N-terminus and his-tagged at the C-terminus) was similarly prepared in Sf9 cells^{46 (link)}. The soluble extract was prepared without phosphatase inhibitors and construct was first bound to amylose resin (New England Biolabs). After elution in MBP elution buffer (50 mM Tris-Cl pH 7.5, 5 mM β-mercaptoethanol, 10% glycerol, 10 mM maltose, 300 mM sodium chloride, 1 mM magnesium chloride), 20'000 units of λ-phosphatase (New England Biolabs) was added to the protein obtained from 1.6 L Sf9 cell culture (approximately 1 mg). The reaction was incubated for 30 min at 30 °C. PreScission protease was added (1:8, w:w) to cleave off the MBP tag, and the solution was incubated for 2 h at 4 °C. The C-terminally His-tagged Sae2 was bound to NiNTA resin (Qiagen), eluted with in a buffer with 300 mM imidazole, and dialyzed into storage buffer (50 mM Tris-HCl pH 7.5, 5 mM β-mercaptoethanol, 10% glycerol, 300 mM sodium chloride), and stored at −80 °C.

The media containing btBDNF were harvested and plates were washed using a washing buffer (30 mM phosphate buffer (pH 8.0), 500 mM NaCl, 20 mM imidazole and a cocktail of protease inhibitors) (Sigma-Aldrich). The media were then incubated on ice for 15 min, centrifuged at 18,000 rpm for 30 min using a Beckman JA-20 rotor and the supernatant was collected. Ni-NTA resins (Qiagen, Hilden, Germany) were rinsed with the washing buffer and added to the collected supernatant at a concentration of 0.3 mL Ni-NTA resins to 100 mL media and incubated overnight on a shaker at 4 °C. The media/Ni-NTA slurry was loaded onto a column and the captured Ni-NTA resins were washed with 10 mL wash buffer and eluted with the elution buffer (30 mM phosphate buffer, pH 8.0, 500 mM NaCl, 300 mM imidazole, protease inhibitors). The purity and concentration of BDNF was assessed by SDS-PAGE using a fast silver staining kit (G-Biosciences, St Louis, MO, USA). Known quantities of BDNF and BSA were used as standards. To precipitate proteins with trichloroacetic acid (TCA), 100% ice-cold TCA was added to the supernatant at a final concentration of 5–7% and incubated on ice for 20 min. The sample was then centrifuged for 20 min at 14,000 rpm. The pellet was washed three times with acetone, air-dried and heated in an SDS loading buffer.