Protein g column

Ascite was filtered through a 0.45 μm syringe filter and 5-fold diluted with PBS, before passing the fluid through protein L or protein G columns (GenScript, Piscataway, NJ, USA). The column resins were washed 3 times with PBS, and immunoglobulins were eluted with 0.1 M glycine (pH 3.0) buffer and neutralized by addition of 1M Tris-HCl (pH 8.0). The purified monoclonal antibodies were further diluted with PBS and analyzed by bicinchoninic acid (Sigma) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) assays by comparing them to mouse IgG (Abcam, Cambridge, UK).

First, total immunoglobulin was obtained in the serum of RA patients and healthy controls; then IgG was purified based on a previously reported method [53 (link)]. Briefly, serum (500 μL) separated from each sample by centrifugation was diluted with phosphate-buffered saline (PBS) (1 mL, pH 7.4), and then 1.5 mL of saturated ammonium sulfate solution was added dropwise and mixed by vortex. The achromatous emulsion was centrifuged, and the deposit was dissolved in PBS (200 μL, pH 7.4) as total immunoglobulin. The collected immunoglobulin was used for IgG purification. The IgG was collected by using protein G columns (GenScript, Nanjing, China). The concentration of proteins was determined using the bicinchoninic acid assay kit (Beyotime biotechnology, Nanjing, China), and the purity was identified by SDS-PAGE.

Embryos and larval tissues were stained with standard immunohistochemical procedures. The following antibodies were used: rabbit anti-V5 (1:1000, Invitrogen); mouse anti-GFP (1:1000, Invitrogen); rabbit-anti-Sas^FL (1:2000, gift of D. Cavener); rat-anti-Sas^short (1:50, GenScript USA Inc); mAb Cq4 against crumbs (1:100, DSHB); guinea pig-anti-Numb (1:1000, gift from J. Skeath); rabbit-anti-dArc1 (1:100, gift from T. Thomson); mAb 8B2 against Ptp10D (1:5, DSHB); mAb MR1A against Prospero (1:40, DSHB); rat-anti-Repo (1/2000, gift from S. Banerjee); rabbit anti-Evi (Wntless, 1:5000, gift from K. Basler); FITC-conjugated phalloidin (1:1000, Thermo Fisher Scientific); AlexaFluor 488 anti-mouse, AlexaFluor 488 anti-rat, AlexaFluor 568 anti-rabbit, AlexaFluor 568 anti-rat and AlexaFluor 647 anti-mouse (1:1000, Invitrogen). Rat anti-Sas^short antibody was generated against a synthetic peptide, HSSIPANGANNLQP, flanking the EVT region (intron is between the N and G residues) and the KLH-conjugated antibody was purified by protein G column (GenScript USA Inc). Samples were mounted in VECTASHIELD (Vector Laboratories) and analyzed on a Zeiss LSM 880.