Pierce protein a agarose

Similar to the method for expressing GPs^{52 (link)}, pcDNA 3.1(−) expression vectors containing encoding gene for different anti-Nipah mAbs was transiently transfected into HEK 293-F FreeStyle cells. After seven days, supernatant was harvested, filtered, and purified using Pierce ProteinA Agarose (Thermo Fisher Scientific). The beads were washed with PBS and the bound protein was eluted with 0.1 M glycine pH 2.0 and neutralized with 1 M Tris pH 8.0. Fractions containing antibody were collected and buffer exchanged into PBS. Proteins were assessed using NuPAGE 4–12% Bis–Tris Protein Gels (Invitrogen) followed by InstantBlue Protein Stain (Expedeon) to confirm purity.

Antibodies: mouse anti-Cx43 IF1 (that binds aa 360–382, from Paul Lampe lab, see [55 (link)]), mouse anti-Cx43 252–270 (m252-270, Cat. # 610061; BD biosciences), mouse anti-actin (Cat. #MA1-744; Thermo scientific, Rockford, IL), rabbit anti-GST (Cat. # G7781; Sigma-Aldrich, St. Louis, MO). Pierce protein A agarose (Cat. # 20333; Thermo Scientific, Rockford, IL) beads were washed with 1% Bovine Serum Albumin (BSA) in 1X PBS and 10% SDS. The agarose beads were then washed three-four times in 1X PBS to remove the SDS and binding solution was added (1% BSA in 1X PBS). 4 μl of the antibody was added to the agarose beads. The antibody was allowed to bind to the resin at 4°C for 4 hours. After incubation, the agarose beads were washed four times with cold 1X PBS. More binding solution and 50 μg (for the overloading condition) or 20 μg (for the standard condition) of bait protein (CT fragments or deletions or F-actin) and prey protein (tubulin, GST-ZO-1^PDZ2, Drb^1-300, and transferrin) were added to the agarose beads. After overnight incubation, the agarose beads were washed four times with cold 1X PBS and 20 μl of 5% BME Novex Tricine loading sample buffer (Life Technologies, Inc., Carlsbad, CA) was added to each sample.

Antibodies against tubulin (Sigma-Aldrich, St. Louis, MO, USA), histone H3 and SUMO1 (Abcam, Cambridge, MA, USA), p65 (Millipore, Darmstadt, Germany), rabbit IgG (Cell Signaling Technology, Beverly, MA, USA), Alexa Fluor 488-conjugated and 568-conjugated IgG (Invitrogen, Carls-bad, CA, USA) were used. Lipofectamine 2000 and Opti-MEM were obtained from Invitrogen (Carls-bad, CA, USA). N-Ethylmaleimide (NEM) was purchase from Sigma-Aldrich (St. Louis, MO, USA). TNF-α was obtained from R & D Systems (Minneapolis, MN, USA). Pierce Protein A agarose was obtained from Thermo (Rockford, IL61001, USA). The dual-luciferase reporter assay kit was obtained from Promega (Madison, WI, USA).

Human sera were obtained from anonymized adult human volunteers. Mouse immune sera were generated by 3x i.p. SA infection with 3.5 × 10⁷ CFU, or 3x immunization with 60μg IsdB adjuvanted with 600μg alum as previously described.¹³ (link) Total mouse IgG were purified from naive and immune mouse sera using Pierce Protein G Agarose (ThermoFisher Scientific), and total human IgG were purified from human sera using Pierce Protein A Agarose (ThermoFisher Scientific). Antigen-specific antibodies were purified from immune mouse or human sera using recombinant proteins immobilized in NHS-activated agarose columns (ThermoFisher Scientific). An example of the purity of human and mouse purified antibodies is shown in Figure S2B.

Functional monomeric and polymeric scEDIII-PIGS were purified at a small scale using Pierce Protein A agarose (Thermo Fisher Scientific Inc.). Eluent fractions were collected in 1 ml each and subsequently concentrated with either 5 × (v/v) cold acetone or Amicon Ultra-15 centrifugal filter (Merck Millipore). Alternatively, the proteins were purified with Bioneer’s Maglisto magnetic protein A kit. The eluents were analyzed on 8% (v/v) SDS-PAGE gel in a non-denatured condition (without boiling in non-reducing 6 × loading buffer) and the gel was blotted on a Nylon membrane. Purified and active scEDIII-PIGS were detected as described above.