ab45977 stained in HL60 cells. Cells were fixed with 100% methanol (5min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab45977 at 5µg/ml and ab72791 (Mouse monoclonal to alpha Tubulin - Loading Control) at 1ug/ml overnight at +4°C. The secondary antibodies used were ab150081 used at 1 ug/ml (colored green) and ab150116 (pseudo-colored red) used at 2ug/ml for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature.

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This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab45977 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406

The mature myeloperoxidase protein is a tetramer of two heavy chains (60 kDa) and two light chains (12 kDa).  Our immunogen sequence is within the myeloperoxidase light chain.  In HL60 cells, ab45977 detects bands at approximately 84-kDa, corresponding to the expected MW of full-length Myeloperoxidase protein, and at 12-kDa, corresponding to the expected MW of myeloperoxidase light chain.