1 research outputs found
Direct Detection of Collagenous Proteins by Fluorescently Labeled Collagen Mimetic Peptides
Although fibrous collagens are major structural components
of extracellular
matrix in mammals, collagen overproduction is associated with many
human diseases including cancers and fibrosis. Collagen is typically
identified in biomedical research by Western blot and immunohistochemistry;
however, anticollagen antibodies employed in these analyses are difficult
to prepare and their affinities to collagen can diminish if collagen
becomes denatured during analyses. Previously, we discovered that
single-stranded collagen mimetic peptides [CMPs, sequence: (GlyProHyp)<sub>9</sub>] can bind to denatured collagen chains by triple helix hybridization.
Here, we present collagen-specific staining methods using simple CMPs
conjugated to common fluorophores (e.g., carboxyfluorescein), which
allow direct detection of collagens and collagen-like proteins in
SDS-PAGE and in various mammalian tissue sections. By directly staining
SDS-PAGE gels with fluorescently labeled CMPs, both intact (type I,
II, and IV) and MMP-1 cleaved collagen (type I) chains as well as
complement factor C1q were detected. Collagen bands containing as
little as 5 ng were optically visualized, while no staining was observed
for fibronectin, laminin, and a collection of proteins from mammalian
cell lysate. The CMP was unable to stain collagen-like bacterial protein,
which contains numerous charged amino acids that are believed to stabilize
triple helix in place of Hyp. We also show that fluorescently labeled
CMPs can specifically visualize collagens in fixed tissue sections
(e.g., skin, cornea, and bone) more effectively than anticollagen
I antibody, and allow facile identification of pathologic conditions
in fibrotic liver tissues