Title: Collagen XII in Fibromodulin-null Mice
Ellen Stice (Presenter)
Texas A&M College of Dentistry
Teniola Omopariola, Texas A&M College of Dentistry
Georgia Mitchell, Texas A&M University College of Dentistry
Reginald Taylor, Baylor College of Dentistry
Objectives: Collagen XII is a protein associated with Collagen I, the most abundant collagen in the human body. Fibromodulin assists collagen assembly in the extracellular matrix. By assessing the relationship between fibromodulin and collagen XII, knowledge can be gained about the organization of the fibers in the skin. This study investigates the presence of collagen XII in the fibromodulin-null mice.
A dot blot immunoassay was performed to compare the presence of collagen type XII in fibromodulin-null mice (KO), wild type mice, and type XII antigen. Tissue samples were snap frozen with liquid nitrogen and stored at -80°C. Protein was extracted using a low salt buffer. The samples were quantified with a BCA standardization kit, and diluted to be within the linear working range. Equal amounts (0.26 nanograms) of protein sample were loaded onto a nitrocellulose membrane, and allowed to dry. Membrane was blocked with a 4% BSA solution for 1 hour and incubated with rabbit anti-collagen XII primary antibody at 4°C for 48 hours. The membrane was processed with a goat anti-rabbit secondary antibody for 1 hour at room temperature, followed by an avidin-biotin complex kit for peroxidase detection. 3,3’-diaminobenzidine (DAB) peroxidase substrate was used for colorimetric detection. LI-COR Image Studio™ Lite software was used for image processing and data collection. Appropriate statistical tests were performed.
The signal intensity is lower in the knock-out fibromodulin mice when compared to the wild-type. This is not statistically significant.
There is probably a decrease in Type XII collagen in the knock-out samples but it is not statistically significant. This could be due to the number of samples. More experiments would be helpful to assess the full extent of the effects of the lack of fibromodulin on collagen XII expression.