Figure 7
Detection and isolation of HAPs in human HIV+ peripheral blood lymphocytes (PBL) with antibody to an internal HAP peptide (4041). (A) Patients HIV (+) PBL 2–5 or HIV (-) PBL-1 cell lysates were fractionated by 4–20% tris-glycine SDS-PAGE and analyzed by Western blot with biotin-labeled rabbit anti-KLH antibody (B-KLH Ab) and with biotin-labeled rabbit anti-4041 antibody (B-4041 Ab). M, MultiMark multi-colored standard. (B) Cell lysates from HIV (+) PBL 5 were analyzed in replicate on 4–20% tris-glycine SDS-PAGE followed by Western blot using biotin-labelled rabbit anti-4041 Ab, or rabbit anti-p24 Ab, HIV Ig (labeled AIDS) and normal human sera, followed by biotin-labelled anti-rabbit Ig or anti-human Ig. (C) HIV (+) PBL 5 lysate was fractionated in the presence of 2 ME (as reducing agent) by 4–20% tris-glycine SDS-PAGE, transferred to membrane and probed with biotin-labeled anti-4041 Ab in the presence (+) or absence (-) of normal rabbit serum. M, MultiMark multi-colored standard. (D) The specificity of the anti-4041 Ab is demonstrated on immunodetection of the + peptide (4041), but not an unrelated peptide (4040, -). In the left panel, the anti-4041 Ab is used in immunoblotting detection of the peptides run in the gel and transferred to membrane. In the right panel, the anti-4041 Ab (lane 1) or unrelated antibodies (lanes 2,3) are separated on SDS-PAGE, transferred to membrane, and then probed with the biotin-labeled peptides, as indicated. (E) Cell lysates from HIV (-) PBL 1 were analyzed in replicate on SDS-PAGE followed by Western blot using rabbit anti-4041 Ab, rabbit anti-p24 Ab, HIV Ig (labeled AIDS) and normal human sera, as in (B). (F) Affinity-isolation of human (HIV +) PBL 5 cell lysates with HAP peptide 4041-specific antibody linked matrix. The affinity-purified anti-4041 Ab was cross-linked to coupling gel with 5 M sodium cyanoborohydride, as instructed (Pierce). Patient PBL 5 cell lysate (60 microliters) was incubated, in duplicate samples, with the anti-HAP peptide (4041) Ab linked gel in a spin column for 2 hours at RT, then the columns washed four times, and bound proteins eluted, as recommended (Pierce, Profound Mammalian Co-Immunoprecipitation Kit). Eluate fractions 1 and 2 from cell lysate samples that had been untreated (NT) or reduced and alkylated (D/I) were run in replicate on the same 4–20% tris-glycine SDS-PAGE. The gel was split along a marker lane (Biorad) and then silver stained (Invitrogen silver staining kit). The mass marker lanes on either end of the gel are MultiMark multi-colored standards (Invitrogen). Molecular weights are in kilodaltons.