Irdye 800cw conjugated goat anti rabbit igg antibody

Mouse anti-puromycin antibody (PMY-2A4) was obtained from Developmental Studies Hybridoma Bank and deposited to the Developmental Studies Hybridoma Bank by Jonathan Yewdell. Rabbit mAb against tubulin, rabbit polyclonal antibody against GFP, and mouse mAb against the His₆ tag were purchased from Cell Signaling Technology (2125), Millipore/Sigma (G1544), and GenScript (A00186), respectively. HRP-conjugated goat anti-mouse IgG and anti-rabbit IgG antibodies were obtained from Bio-Rad (170-6516 and 170-6515, respectively). IRDye® 800CW–conjugated goat anti-rabbit IgG antibody and IRDye® 680LT-conjugated goat anti-mouse IgG antibody were purchased from LI-COR (926-32211 and 926-68020, respectively).

Western blotting was performed using routine methods. Primary antibodies against the following proteins were used: mouse anti-GAPDH (1:5,000; Invitrogen MA5-15738), and rabbit anti-A1 AR (1:200; Alomone AAR-006). Secondary antibodies were used: IRDye 800CW-conjugated goat anti-rabbit IgG antibody (1:5,000; Li-Cor 926–32211), or IRDye® 680RD-conjugated goat anti-mouse IgG antibody (1:5,000; Li-Cor 926–68070). Protein bands were visualized using an Odyssey Infrared Imaging System (Li-Cor Biosciences). Details are provided in the Supplementary Methods.

The following primary antibodies were used: rabbit polyclonal anti-LEDGF/p75 (Bethyl Laboratories; product code A300-848A), rabbit polyclonal anti-HRP2 (Novus Bio; product code NBP2-47438), affinity-purified polyclonal rabbit anti-MVV capsid/p24 antibody (Thermo Fisher Scientific, custom product; see Supplementary Fig. 12 for validation data), and horseradish peroxidase-conjugated rabbit monoclonal anti-β-actin antibody clone 13E5 (Cell Signaling Technology, product code 5125). Blots probed with anti-LEDGF/p75 (diluted 1:5000 in phosphate-buffered saline, supplemented with 0.05% Tween-20 (PBST)), anti-HRP2 (diluted 1:1000 in PBST), and anti-capsid/p24 antibodies (1:5000) were developed following incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (BioRad; product code 1706515; diluted 1:10,000 in PBST) or IRDye 800CW conjugated goat anti-rabbit IgG antibody (LI-COR; product code 925-32211; diluted 1:2000 in PBST) for detection by chemiluminescence, using Imager 600 RGB (GE Healthcare) following incubation with ECL prime reagent (GE Healthcare) or by fluorescence, using an Odyssey imager (LI-COR), respectively.