Generation of Schlafen 8-Specific Antibodies.

Abstract

Background/Objectives: Schlafen (SLFN) 8 and SLFN9 are mouse members of the Schlafen protein family, believed to have arisen through a gene duplication event. The physiological roles of these proteins remain poorly defined, in part due to the absence of reliable, commercially available antibodies for their detection. Methods: To develop specific antibodies, we performed an amino acid sequence alignment of these proteins and identified a thirteen amino acids long peptide predicted by AlphaFold modeling and hydropathicity analysis to be surface-exposed in both SLFN proteins. The SLFN8 peptide was conjugated to KLH and used to immunize mice, employing Poly(I:C) as an adjuvant. Results: We verified the anti-SLFN8 antibody specificity in mouse tissues, engineered human cells, and recombinant proteins by different immunodetection techniques, including Western blotting, immunoprecipitation, immunohistochemistry, and ELISA. Furthermore, splenocytes from immunized mice were used to generate hybridomas that secreted IgG antibodies with SLFN8-peptide specificity, as assumed by ELISA. Conclusions: Our results demonstrate that the identified peptide is highly immunogenic and capable of eliciting antibodies that distinguish between these two exceedingly similar proteins in a broad group of immunodetection techniques.