Alexa flour 488 conjugated anti mouse igg

MCF7 cells were seeded onto coverslips in 6-well plates and cultured for an additional 24 h. Cells were treated with either DMSO or 10 μM RU-SKI 43 for 4 h. Cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.2% Triton X-100 for 5 min at room temperature. Cells were incubated with anti-ERα (Cell Signaling) for 1 h followed with incubation with a secondary antibody (Alexa Flour® 488-conjugated anti-mouse IgG) for 45 min. Slides were mounted with ProLong® Gold Antifade (Invitrogen). Images were collected using a Leica SP5 confocal microscope and analyzed with the Leica Application Suite software. Images were collected using the same conditions on the same day ensuring fair side-by-side comparison.

MCF7 or BT-474 cells (2×10⁵) were suspended in 100 µg of serially diluted TrMab-6 (6 ng/ml-100 µg/ml) for 30 min at 4°C, followed by the addition of Alexa Flour 488-conjugated anti-mouse IgG (1:200; Cell Signaling Technologies, Inc.). Fluorescence data were collected using a cell analyzer (EC800). The dissociation constant (K_D) was calculated by fitting the binding isotherms to built-in, one-site binding models in GraphPad Prism 8 (GraphPad Software, Inc.).