Western lightning plus ecl enhanced luminol reagent

For detection of proteins by Western blotting, cells were lysed in sample buffer (50 mM HEPES pH 7.4, 2% SDS, 10% Glycerol, 100 mM DTT). After separation on a SDS/5–20% polyacrylamide gradient gel and transfer to a 0.2 µm pore size nitrocellulose membrane (Peqlab), membranes were blocked in PBS containing 0.1% sodium azide and 5% bovine serum albumin (for IER3) or milk powder. Proteins were detected with the following antibodies diluted in PBS: ZFP36 (Carp3, abcam, ab36558-200), IER3 (Santa Cruz, sc-8454) and EIF3B (Santa Cruz, sc-16377), as well as HRP-coupled anti-goat (Santa Cruz, sc-2020) or anti-rabbit (Jackson ImmunoResearch, 711-036-152) antibody. Between the antibody incubation steps, membranes were washed in 150 mM NaCl, 50 mM Tris-HCl (pH 7.5 at 25°C), 1% Tween-20. As a luminol reagent, Western Lightning Plus-ECL Enhanced Luminol Reagent (Perkin Elmer) was used.

For total protein extracts, cells were washed once with PBS, lysed by directly adding 2× SDS sample buffer, and incubated for 5 min at 95°C. For soluble protein extracts, cells were lysed in RIPA buffer (50 mM Tris–HCl, pH 7.4, 1% NP‐40, 0.25% Na‐deoxycholate, 150 mM NaCl, and 1 mM EDTA) containing protease inhibitors (cOmplete tablets, Roche) and phosphatase inhibitors (50 mM NaF, 40 nM okadaic acid, and 1 mM Na‐vanadate). Samples were resolved by SDS–PAGE, blotted onto 0.2‐μm‐pore‐size nitrocellulose membrane (Peqlab), and blocked in PBS containing 5% milk. HPR‐coupled secondary antibodies were purchased from Santa Cruz and Jackson ImmunoResearch, and Western Lightning Plus‐ECL Enhanced Luminol Reagent (Perkin Elmer) was used as chemiluminescence substrate.

Env proteins were detected by SDS-PAGE, BN-PAGE and Western blotting, as previously described [7 (link),25 (link)]. Briefly, Western blotting was performed after transfer of proteins onto a nitrocellulose membrane (Invitrogen, Carlsbad, CA, USA) at 32–40 V for 1–1.5 h. The membranes were blocked with 10% goat serum for 1 h at room temperature and incubated overnight with a 1:2000 dilution of the anti-gp120 MAb ARP 3119 (obtained from the NIBSC Reagent Repository), followed by a 1:5000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG (H + L) and the Western Lightning plus-ECL Enhanced Luminol Reagent (Perkin Elmer, Waltham, MA, USA). Alternatively, the membranes were probed with a HIV-Ig pool derived from individuals infected with viruses from subtypes A, B and C. The subtype A serum was obtained from Stephanie Rainwater (The Fred Hutchinson Cancer Research Center), the subtype C serum from Zdenek Hel (University of Alabama, Birmingham), and the subtype B serum from the AIDS Research and Reference Reagent Program (ARRRP) (product #3957, lot 110180). Each individual serum was present at a 1:3000 dilution in 10 ml of TBS + 0.05% Tween-20. The detection antibody was a 1:3000 dilution of biotin-conjugated goat anti-human IgG, followed by an Avidin-Biotin-HRP complex and a peroxidase substrate kit (all from Vector Laboratories, Burlingame, CA).