Donkey anti rabbit hrp

Samples were loaded onto a 4-12% Bis-Tris NuPAGE gels and then transferred to nitrocellulose. Transfer was verified by Ponceau staining (0.5% Ponceau Red-S, 2% Acetic acid in H₂O). Membranes were blocked in TBST (50mM Tris-HCl, pH 7.5, 150mM NaCl, 0.1% Tween-20) with 5% powdered milk (blocking buffer) for 1 hour, incubated with primary antibody diluted in blocking buffer for 1h, and washed in TBST. Membranes were exposed to the appropriate secondary antibody diluted in blocking buffer for 1 hour, and washed in TBST prior to incubation with Bio-Rad Clarity reagent and exposure to film. Films were scanned in using a Canon CanoScan 9000F with a transparency adaptor. MYO19 was detected in HeLa cells using 0.3 μg/ml chicken anti-human MYO19 [Quintero et al. 2009 (link)] and 80ng/ml donkey anti-chicken HRP (Jackson Immuno Research). MYO19 was detected in B16F1 samples using 0.17 μg/ml rabbit anti-mouse MYO19 [Rohn et al. 2014 (link)] and 40ng/ml donkey anti-rabbit HRP (Jackson Immuno Research). Porin was detected in all samples using 5ng/ml mouse anti-human porin (LifeTechnologies, A31855), and 10 ng/ml donkey anti-mouse HRP (Jackson Immuno Research).

One-cell Xenopus embryos were injected with capped mRNA for Xenopus transcripts: 5′ myc-neurog2 (50 pg per embryo), vxn (1 ng), and p27Xic1 (50 pg). Embryos were lysed at stage 15 in lysis buffer (20mM Tris-HCl pH 7.4, 140mM NaCl, 1% NP-40, 10% glycerol, 2mM EDTA, 1mM MgCl2, Halt protease inhibitor (Thermo Scientific)), and cleared of debris by centrifugation at 12,000 rpm for 15 minutes. Lysates were run on a western blot and Myc-Neurog2 protein levels were detected with goat anti-c-myc (1:1000, Santa Cruz Biotechnology, sc-789-G), followed by donkey anti-goat HRP (Jackson ImmunoResearch Laboratories) and developed with Clarity Western ECL substrate (BioRad). The membrane was stripped and probed with Rabbit anti-β-actin (1:1000, Abcam), followed by donkey anti-rabbit HRP (Jackson ImmunoResearch Laboratories) as a loading control. Band intensities were quantified with ImageJ, and Myc-Neurog2 protein levels were normalized to β-actin.

Protein expression and immunoprecipitation using anti-FLAG M2 or Myc (9E10) agarose beads (Sigma) were performed as described (Ausubel et al., 2003 ). Primary antibodies used included rabbit anti-FLAG (Sigma, 1:2000), rabbit anti-Myc (Sigma, 1:2000) and rabbit anti-GFP (Santa Cruz, 1:2000). Secondary antibodies used included donkey anti-rabbit HRP (Jackson ImmunoResearch, 1:4000) or IRDye 800CW goat anti-rabbit (Odyssey LI-COR Biosciences, 1:10000).