Irdye680lt and irdye800 conjugated anti rabbit and anti mouse igg secondary antibodies

Worm lysates were generated from unmated fog-2(q71) worms to eliminate contribution from embryos and were resolved on 12% SDS-PAGE gels and transferred to Millipore Immobilon-P PVDF membranes. Membranes were blocked with 5% BSA and probed with rabbit anti-CENPA (1:250, Novus), and anti-Mortalin/Grp75 mouse monoclonal antibody N52A/42 (1:20, AB_10674108; UC Davis/NIH NeuroMab Facility, Davis, CA) as loading control., followed by IRDye680LT- and IRDye800-conjugated anti-rabbit and anti-mouse IgG secondary antibodies obtained from LI-COR Bioscience (Lincoln, NE). 0.01% SDS was added to anti-rabbit secondary.

Whole worm lysates were generated from indicated worms; unmated fog-2(q71) worms were used to eliminate embryos. ~100 worms were collected, washed in M9 buffer and resuspended in equal volume of 2X Laemmli sample buffer (Bio-RAD). Lysates were resolved on 4–15% SDS-PAGE gradient gels (Bio-RAD) and transferred to Millipore Immobilon-P PVDF membranes. Membranes were blocked with 5% nonfat milk and probed with rabbit anti-GFP (1:1000; NB600-308; Novus Biologicals, Littleton, CO) and mouse anti-α-tubulin (1:1000; Sigma-Aldrich; T9026) as loading control, followed by IRDye680LT- and IRDye800-conjugated anti-rabbit and anti-mouse IgG secondary antibodies (1:20000; LI-COR Bioscience Lincoln, NE). Immunoblots were imaged on a LI-COR Odyssey Infrared Imager, signal was quantified using Fiji and normalized with the α-tubulin signal.