Rat anti rfp antibody

For yeast, 1 OD₆₀₀ of mid-log phase cells was collected by centrifugation and precipitated using 10% trichloroacetic acid for 20 min at 4 °C. After centrifugation at 13,000g for 5 min, pellets were washed with ice-cold acetone. Pellets were air-dried and resuspended in 30 µl of 1× SDS sample buffer (60 mM Tris pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol and 0.005% bromophenol blue), and boiled for 3 min. For mammalian cells, 10⁶ cells were scraped off in 100 µl of SDS sample buffer and heated at 96 °C for 10 min. Samples were resolved on a 12% SDS–PAGE gel, and after transfer on a PVDF membrane, proteins were detected using specific antibodies. The following antibodies were used: mouse anti-Pgk1 antibody (Invitrogen, 459250, 1:3,000 dilution), rat anti-RFP antibody (Chromotek, 5F8, 1:1,000 dilution), mouse anti-FLAG antibody (Sigma, F1804, 1:1,000), rabbit anti-mCherry antibody (Abcam, ab167453, 1:1,000) and horseradish-peroxidase-coupled secondary antibody (Bio-Rad, 170–6516; 1:10,000 dilution). Western blots were imaged using the Fusion FX system (Vilber) equipped with the FusionCapt Advance FX7 software (version 17.03).

Larval imaginal discs were stained according to Papadopoulos et al. (2010) (link). Staining for the endogenous Antp protein were performed using a mouse anti-Antp antibody (Developmental Studies Hybridoma Bank, University of Iowa, anti-Antp 4C3) in a dilution of 1:250 for embryos and 1:500 for imaginal discs. eGFP, or eGFP-tagged proteins, were stained using mouse or rabbit anti-GFP antibodies from Thermo Fisher Scientific at 1:500 in imaginal discs and 1:250 in embryos. mRFP1 was stained using a Chromotek rat anti-RFP antibody. For Antp P1 promoter staining in imaginal discs, we used the mouse anti-β-galactosidase 40-1a antibody from Developmental Studies Hybridoma Bank, University of Iowa at 1:50. The rabbit anti-Scr antibody was used at 1:300 (LeMotte et al., 1989 (link)). Confocal images of antibody staining represent predominantly z-projections and Zeiss LSM510, Zeiss LSM700 or Zeiss LSM880 Airyscan confocal laser-scanning microscopy systems with an inverted stand Axio Observer microscope were used for imaging. Image processing and quantifications have been performed in Fiji (Schindelin et al., 2012 (link)). For optimal spectral separation, secondary antibodies coupled to Alexa405, Alexa488, Alexa594 and Cy5 (Thermo Fisher Scientific) were used.