Illuminating Small Proteins Through HiBiT Blotting.

Epitope tag immunoblotting represents a routine method for targeted surveys of protein abundance and expression. Especially for microproteins, defined by an arbitrary cutoff of 100 amino acids in length, blotting-based approaches are indispensable as the small size of microproteins often renders them elusive to mass spectrometry. Nonetheless, the blotting of microproteins introduces a set of technical challenges, leading to microprotein losses, which significantly affect the sensitivity of blotting-based approaches like epitope tag immunoblotting.We introduce HiBiT blotting, an antibody-free luminescent detection method for HiBiT-tagged proteins, offering an alternative blotting-based protein detection method to improve microprotein analysis. The availability of an anti-HiBiT antibody enabled a comparative analysis of HiBiT versus classical epitope tag immunoblotting, overall demonstrating the superior sensitivity of HiBiT blotting in detecting HiBiT-tagged microproteins. By offering a more direct and sensitive approach for small protein analysis, HiBiT blotting represents a substantial contribution to the field, enabling the effective study of microproteins and addressing the longstanding challenge of their detection.