Alexa fluor 647 streptavidin

U2OS cells were grown on glass coverslips and fixed in 4% paraformaldehyde/PBS for 10 minutes at room temperature, permeabilized for 5 minutes in PBS containing 0.5% Triton X-100 and blocked by incubation in IF blocking buffer (PBS containing 0.5% cold water fish gelatin (Sigma, Vienna, Austria)) for 15 minutes. All primary and secondary antibody dilutions were prepared in IF blocking buffer and applied for 60 minutes. DNA was stained with 1 µg/ml DAPI (Sigma, Vienna, Austria) and coverslips were mounted on microscopy slides in Mowiol (Sigma, Vienna, Austria). All samples were imaged using a confocal laser scanning microscope (LSM-Meta 510, Carl Zeiss AG, Oberkochen, Germany) using a Plan-Apochromat 63x/1.4 Oil DIC objective. Primary antibodies used for immunofluorescence were the same as used for Western Blotting. In addition, an Alexa Fluor™ 647-Streptavidin (016-600-084, Jackson ImmunoResearch, West Grove, Pennsylvania, USA) was used for detection of biotinylated proteins. Secondary antibodies conjugated to Alexa-488 were purchased from Molecular Probes and secondary antibodies conjugated to Cy5 or TexasRed from Jackson ImmunoResearch (West Grove, Pennsylvania, USA).

HEK-293T cells were transfected with viral gene vectors, each which encode in-frame 2x Strep tag fusion as described in [42 (link)]. Twenty-four hours post transfection, cells were collected and washed with PBS containing 0.5% BSA, before fixation with 2% PFA in PBS for 15 minutes at RT. After this, cells were washed twice, then permeabilized using ice cold methanol for 30 minutes at -20°C. Cells were then washed before incubation with anti-Strep antibody for 1h on ice, according to the manufacture’s protocol (IBA, strepMAB-Classic 2-1507-001), washed and incubated with biotinylated anti-mouse antibody for 1 hour on ice. Finally, cells were washed and incubated with Alexa Fluor 647 streptavidin (Jackson Immuno Research, AB_2341101) for 15 minutes on ice and were subjected to FACS analysis.