The staphylococcal collagen adhesin CNA35 effectively detects collagen and its fragments in blots after SDS-PAGE

Abstract

Collagens are a diverse family of proteins present in the extracellular matrix (ECM) of all animals. They play crucial roles in providing structural support to tissues, forming scaffolds for ECM suprastructures, and signaling cells. Certain collagen-binding proteins from pathogenic bacteria, such as CollageN Adhesin (CNA) from Staphylococcus aureus, interact with the collagen triple helix to promote host invasion. The extracellular portion of CNA, known as CNA35, which has a molecular weight of 35 kDa, has been used for in vitro, ex vivo, and in vivo staining of various collagen types in tissues.

Detecting various types of collagens necessitates the use of type- and specie-specific antibodies, which typically exhibit weak affinities for the triple helical regions of collagens. Additionally, the fragmentation of collagens can lead to a loss of detection due to the limited number of available epitopes. Furthermore, antibodies can be expensive, require secondary identification methods, and are often suitable for either immunohistochemistry or western blotting. Although successful procedures for staining collagens in tissues have been implemented, the detection of collagens and their fragments using CNA35 has not been reported for protein blots.

In this study, we examined the detection capabilities of a trimeric form of CNA35 for protein blots following SDS-PAGE. We successfully tested collagens I through VI, as well as fragments of collagen IV, under various conditions. Additionally, we investigated the impact of blocking solutions, incubation time, ligand concentration, and CNA35 concentration on sensitivity.

We achieved superior detection of all tested collagens and collagen IV fragments, including the 7S domain, which is a highly crosslinked complex composed of four triple-helical strands. The method we developed serves as a universal tool for detecting collagens and collagen-containing peptides in protein blots. It offers several advantages, including sub-nanogram sensitivity, low cost, and compatibility with standard western blotting techniques.