Amicon ultra column

The Mtb Alr enzyme was expressed as the short variant characterized by Strych et al. that features a 24-residue N-terminal truncation relative to the annotated Rv3423c gene^{23 (link)}. The enzyme was His-tagged and expressed in the TESEC Alr- Host strain, which has no other source of Alr activity.
The TESEC Alr Purification strain was grown to saturation in 500 mL of LB supplemented with ampicillin, kanamycin, D-alanine, and 100 mM L-arabinose. A cell pellet of approximately 2 grams was harvested by centrifugation, cooled to 4 °C and lysed with 10 mL B-PER reagent (ThermoFisher 78248) and a standard EDTA-free protease inhibitor cocktail (Merck 11873580001).
The lysate was cleared by centrifugation and passed over 3 mL HisPur spin columns (ThermoFisher 88226). Elution fractions were collected with increasing concentrations of imidazole (Sigma I2399) and checked for the presence of a single band using SDS-PAGE gels (Bio-Rad 4561081) and Coomassie staining. Successful elutions were combined and dialyzed (ThermoFisher 66380) against 20 mM Tris, 100 mM NaCl pH 8.0 for 72 h at 4 °C. Finally, samples were concentrated by centrifugal filtration in Amicon Ultra columns (Merck UFC9010). Total protein concentrations were determined using the Rapid Gold BCA Protein Assay (ThermoFisher A53226) calibrated to a standard curve following the manufacturer’s protocol.

Lentiviral vectors containing shRNAs were used to downregulate the expression of each REEP (Thermo Scientific Inc., Waltham, MA) following transfection into HEK293 cells with pcDNA3.1mychis/REEP plasmids. The shRNA constructs were inserted into the lentiviral FG12 vector and transfected into HEK293T cells with accessory plasmids using a calcium phosphate precipitation method. Two days later, the culture supernatants were harvested and concentrated using Amicon Ultra columns (Millipore, Billerica, MA). An EGFP-expressing lentivirus served as a control to evaluate infection efficiency. The multiplicity of infection was 10, and the expression of target proteins was determined by western blotting.

Cells were cultured in serum-free DMEM for 24h. Media was collected and concentrated at 4°C by ultrafiltration using Amicon Ultra columns (Millipore). Media equivalent to 30x10³ cells was run on 10% gelatin zymography gels (Bio-Rad Laboratories, Hercules, CA). Gels were washed in 2.5% Triton X-100 three times 10min each and incubated overnight in developing buffer (50 mM Tris pH 8.2, 5 mM CaCl₂, 0.5 μM ZnCl₂) at 37°C, followed by staining with Coomassie brilliant blue and destaining. Gel images were acquired using Scan Maker i900 (Microtek, Industrial Park Hsinchu, Taiwan).

HEK293T cells cultured in Dulbecco modified Eagle medium (Fisher) with 10% fetal bovine serum (Fisher) were transfected with the Reelin or R3-6 cDNA constructs (Jossin et al., 2004 (link)), using Polyjet (Tebu-Bio). After 24 hr, the medium was replaced with a serum-free medium, which was collected 2 days later and stored at 4°C in the presence of a protease inhibitor cocktail (Complete, Roche). Prior to use, the supernatants were concentrated using Amicon Ultra columns with 100,000-molecular weight cutoff filters (Millipore) to reach the approximate concentration of 400 pM, which was estimated as described previously (Jossin et al., 2004 (link)), and dialyzed against culture medium by drop dialysis (Millipore VSWP02500). Mock solutions were prepared from control transfected HEK293T cells and used to control for potential co-purifying proteins.