Figure 9.
Sypro Ruby and immunoblots (Western) SDS–PAGE gels compare protein standard HCRLC and proteins extracted from adult or embryonic zebrafish. Immunoblot primary antibodies are for RLC (HCRLC and ZRLC) or β-actin. TgGFP(+/−) embryo samples are denoted by GFP(+/−) or G(+/−). Panel a shows partially purified adult WT zebrafish myosin (WT Zmys) and in vitro expressed HCRLC. The latter has concentration measured with the Bradford assay (Biorad, Hercules CA). Sypro Ruby staining of both samples determines the ZRLC content in the WT Zmys sample. The Western of the same samples calibrates immunoblot intensities for HCRLC and ZRLC when using the primary RLC antibody. Panel b shows the immunoblots for purified skeletal myosin from WT, TgGFP(−) and TgGFP(+) zebrafish embryos detecting the unknown expression levels of HCRLC-GFP and ZRLC. Relative mole fraction of HCRLC-GFP (ZRLCrep) is computed using equation (2.2) in the text. A second experiment with the purified myosin provided a ZRLCrep = 0.25 (data not shown). Panel c indicates Western blots from an embryo protein extract detecting HCRLC and β-actin. ZRLC intensities are normalized relative to β-actin expression and compared across the WT, TgGFP(−) and TgGFP(+) zebrafish. The relative intensities measure the loss of ZRLC coincident with gain in HCRLC-GFP expression due to the transgenesis. The ZRLC fraction lost (ZRLCrem) is quantitated using equation (2.3) in the text. Detection of the expressed HCRLC-GFP in the protein extract is also indicated, permitting calculation of ZRLCrep. HCRLC-GFP and β-actin positions in the gel overlap. Panel d indicates Western blots from embryo protein extracts. Like panel c, these samples indicate the relative loss in ZRLC with the gain in HCRLC-GFP expression in the transgenic zebrafish.