Detection of HiBit Tagged Proteins in Zebrafish Embryos (V1 10.05.25) v1

This protocol describes a rapid method for detecting HiBiT-tagged proteins in zebrafish embryos following mRNA microinjection. It outlines construct design, in vitro transcription with capping and polyadenylation, and preparation of a defined microinjection buffer for optimal mRNA delivery. Embryos are lysed by freeze–thaw in E3 medium, followed by the addition of HiBiT lytic reagent to enable quantitative luminescent detection of tagged proteins using the Nano-Glo HiBiT system. The method yields reliable detection of exogenously expressed proteins within hours post-fertilization, requiring only minimal sample processing. Compared with enzymatic or mechanical lysis methods, this approach improves reproducibility, reduces processing time, and preserves HiBiT activity. Version 1 (10.05.25) standardizes freeze–thaw parameters and reagent ratios for zebrafish applications, ensuring compatibility with both early- and late-stage embryos.