Rabbit monoclonal anti human igg clone epr4421

Allophycocyanin (APC)-conjugated mouse anti-human TNF-α (clone MAb11), APC-conjugated mouse IgG1 isotype control (clone P3.6.2.8.1), phycoerythrin (PE)-Cy7-conjugated mouse anti-human CD14 (clone 61D3), PE-Cy7-conjugated mouse IgG1 isotype control (clone P3.6.2.8.1), and brefeldin A were purchased from eBioscience (San Diego, CA). Rabbit monoclonal anti-human SAP (clone EP1018Y) and rabbit monoclonal anti-human IgG (clone EPR4421) were purchased from Abcam (Cambridge, MA). Recombinant human SAP was purchased from BioLegend (San Diego, CA). Human intravenous immunoglobulin G (IgG) was used as a source of purified human IgG and was kindly provided by Grifols Therapeutics (Los Angeles, CA). Ultralow IgG fetal calf serum (FCS) was purchased from Gibco (Waltham, MA). Single-stranded DNA cellulose was acquired from Sigma-Aldrich (St. Louis, MO). A custom Milliplex human cytokine/chemokine magnetic bead kit was purchased from Millipore Sigma (Burlington, MA).

Totally, 10 × 10⁶ Ramos cells or 5 × 10⁶ CHO cells expressing various CD40 mutants were incubated with 20 µg/mL anti-CD40 mAbs for 30 min before being washed three times with PBS and lysed in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% Deoxycholate, 0.1% SDS, 50 mM Tris, pH 8) supplemented with 2 mM Na₃VO₄, 50 mM NaF, and 1× Protease Inhibitor Cocktail (Sigma, UK). Cell lysates were centrifuged and determined for their protein concentration using Bradford assay (Biorad, UK), and then 10 µg cell lysate was reduced and loaded onto 12% Bolt gel for SDS-PAGE (Thermofisher, UK). Proteins were then transferred to an iBlot 2 nitrocellulose membrane using the iBlot 2 Gel Transfer Device (both from Thermofisher) and the membrane was blocked using PBST 5% milk for 30 min. The membrane was then probed overnight at 4 °C with rabbit monoclonal anti-human IgG (clone EPR4421, Abcam, UK, 1/1000) and rabbit anti-β-Actin (clone 13E5, Cell Signalling Technology, UK, 1/1000) before detection using secondary goat anti-rabbit IgG-HRP (Abcam, UK, 1/5000). The membranes were developed using Immobilon Classico Western HRP substrate or Immobilon Western Chemiluminescent HRP Substrate (both from Sigma) and chemo-luminescence captured using the UVP Biospectrum Imaging System.