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Figure 1 | Retrovirology

Figure 1

From: CD45 immunoaffinity depletion of vesicles from Jurkat T cells demonstrates that exosomes contain CD45: no evidence for a distinct exosome/HIV-1 budding pathway

Figure 1

Immunoblots and SDS-PAGE gels of vesicles and virion samples. Immunoblots and SDS-PAGE gels of vesicle preparations (A) or virion preparations (B) (equal amounts by volume) isolated from cell cultures are presented. The samples are identified above their respective lanes. Antibody or antiserum used is indicated. Pertinent bands are identified at right of the blots. Cellular proteins reduced in depleted virion preparations are denoted in panel B with a dot at right. Cells were cultured in RPMI 1640 media with 2 mM L-glutamine, 100 U per ml penicillin, 100 μg per ml streptomycin and 10% vol/vol fetal bovine serum. CD45 immunoaffinity depletion was carried out using 100 μl of anti-CD45 paramagnetic microbeads (cat # 130-045-801, Miltenyi Biotec Inc.) that were washed in PBS and recovered by a magnetic separator (model MPC-S, Invitrogen, Inc.) twice before use. After an hour incubation at room temperature, the suspension was placed in a magnetic separator overnight at 4°C to capture the beads. The supernatant carefully removed from the beads and analyzed. Pan-specific CD45 antibody was obtained from BD-Transduction Laboratories, San Diego, CA, cat # 610266, Clone 69, IgG1. The pan-actin antibody was obtained from Amersham Biosciences, Arlington, IL, cat # N.350. CA antiserum was from the AIDS and Cancer Virus Program, NCI-Frederick, Goat # 81. SDS-PAGE gels were stained with by Coomassie brilliant blue to visualize proteins.

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