Additional file 2.
Cytometric detection of tightly bound proteins. Exponentially growing Molt4 cells were directly fixed in methanol (MeOH) or first treated with Triton X-100, fixed with MeOH (TX-100/MeOH); then stained for Cdc6, ORC2, ORC3, Mcm2, cyclin A2, using indirect staining with FITC conjugated goat anti-mouse antibodies and counter-stained for DNA content (DAPI), then measured by cytometry. In the first two panels (Cdc6), a secondary antibody control was run (blue outlines), demonstrating elevated background in MeOH-fixed cells as expected. Secondary only controls were not run with the remaining antigens. In all cases, a significant loss of signal was achieved after detergent extraction. The results agree quantitatively with Western blots for Cdc6 (Table 1) and agree qualitatively for the other antigens (Additional file 1). The detergent extracted pattern for Mcm2 demonstrates bimodality in G1 (arrows), declining signal in S phase, and a return to baseline in G2+M. This pattern is equivalent to that for Mcm6.
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Frisa and Jacobberger BMC Cell Biology 2010 11:26 doi:10.1186/1471-2121-11-26