HL 2°Abs show a bias for mAbs of different IgG subclasses in a variety of applications.

<p>(A) A single immunoblot containing samples of crude rat brain membranes (RBM, 50 µg protein) and extracts of transfected COS-1 cells expressing individual target proteins, or from control cells transfected with an empty plasmid as labeled, probed with anti-PSD-95 (IgG2a), anti-Kv1.2 (IgG2b) and anti-Kv2.1 (IgG1) mAbs, and HL 2°Ab (green), and a cocktail (1∶1∶1) of SCS anti-IgG1, -IgG2a and -IgG2b 2°Abs (red). Multicolor panel is original immunoblot; single color panels are images of separated colors. Changes in tint reflect bias of HL for (more green) IgG2a>IgG2b>IgG1 (more red). Lane to left shows molecular weight standards in kDa. (B) FLISAs show that IgG subclass bias of HL is present at all concentrations of 1°Abs. Left panel: SCS 2°Abs (each at 1 µg/ml). Right panel: HL 2°Ab. Circles: L76/36 IgG2a; triangles; K14/16 IgG2b; squares: K14/39 IgG1. (C) IgG subclass bias is also present in immunofluorescence labeling of Kv1.2-expressing COS-1 cells. Cells were labeled with mAb as noted, and HL 2°Ab (red), and SCS 2°Abs (green) as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038313#s4" target="_blank">Methods</a>. Changes in red∶green tint reflect bias of HL for (more red) IgG2a>IgG2b>IgG1 (more green). Scale bar = 100 µm. Panel to right is quantitation of immunocytochemistry results from three fields each of three independent samples.