The monoclonal pS62 antibody 33A12E10 recognizes c-Myc but also cross-reacts with a serum protein. (A) pS62 (33A12E10) immunoprecipitates c-Myc. Cells (3×10⁷ JY) were lysed in Ab lysis buffer and incubated overnight at 4°C with either mouse IgG or 33A12E10, followed by protein A beads for 1 hour. Bound protein was washed with Ab lysis buffer, boiled in SDS sample buffer, and separated by SDS-PAGE. Immunoblotting was performed with 33A12E10 and N262 antibodies. Asterisks indicate IgG heavy chain. (B-C) Serine 62 is required for recognition of the pS62 (33A12E10) antibody. The indicated V5-tagged mouse c-Myc constructs were expressed in yeast (B) or 293 cells (C), and lysates were separated by SDS-PAGE. Immunoblotting was performed with 33A12E10 and N262 antibodies. (D) 33A12E10 robustly detects a protein present in trace amounts of FBS. RPMI (10 μL) or RPMI + 10% FBS (10 μL) without cells was resuspended in 100 μL of SDS sample buffer and boiled, and 5 μL were separated by SDS-PAGE and immunoblotted with 33A12E10. (E) Washing cells with PBS reduces cross-reactivity of 33A12E10. One million MV4-11 cells were washed with either 1 mL or 10 mL of PBS, then cells were lysed in SDS sample buffer. Lysate from 2 × 10⁵ cells was separated by SDS-PAGE, and immunoblotting was performed with 33A12E10 and Y69 antibodies. Irrelevant intervening lanes were removed. (F) A form of pS62 c-Myc co-migrates with the cross-reacting serum protein. MOLM-14 cells were harvested with and without 3 sequential PBS washes (1 mL each). Cells were counted before final spin (to control for cells lost during washing), and pellets were lysed in SDS sample buffer. Lysate from 1.5 × 10⁵ cells was separated by SDS-PAGE, and immunoblotting was performed with 33A12E10, C19, and N262 antibodies. For all immunoblots, proteins were separated by SDS-PAGE, transferred to Immobilon-FL membrane, and blocked with Aquablock. Blots were incubated with the indicated primary antibodies followed by goat anti–mouse or anti–rabbit secondary antibodies conjugated to either Alexa Fluor 680 or IRDye800 and imaged on a LI-COR Odyssey scanner. The following primary antibodies were used: pS62 c-Myc (33A12E10; 1:500) and Y69 (1:1000; both from Abcam); N262 (1:1000) and C-19 (1:100; both from Santa Cruz Biotechnology); and GAPDH (1:10 000; Ambion). c-Myc is indicated with a black arrowhead; FBS cross-reacting band is indicated with a gray arrowhead.