#Cell biology lab western blot results full#

Western blot methods are needed to detect changes in collagen type 1 protein synthesis, in which synthesis of intracellular procollagen products might increase more than extracellular mature collagen. Excessive deposition of extracellular matrix is also associated with other disorders, such as pathological mechanical overload of the heart. Thus, the exact parameters employed are dependent upon the collagen protein product that the scientist desires to identify.Ī key goal of our lab is to examine mechanisms underlying overuse-induced muscle, tendon and nerve fibrosis and effective treatments for these pathological tissue changes. Boiling is to be avoided as the antigenic site recognized by the monoclonal antibody used is sensitive to thermal denaturation, as is the case with many monoclonal antibodies available on the market. For characterization of mature collagen 1(I), both 8% and gradients type gels are appropriate, although still without SDS, yet with SDS included in both running and sample buffers, BME must be added to the sample buffer, and samples should not be boiled. Also, BME must not be added to the sample buffer and samples should not be boiled. The best resolution of type 1 procollagens is achieved using 4%–12% Tris-glycine gels without the presence of SDS in the gel itself, although SDS in the running and sample buffers are needed.

#Cell biology lab western blot results full#

Our results indicate full native gel conditions prevent resolution of all collagen type 1 bands. no boiling) and (3) running buffer composition (SDS vs. non-gradient, sodium dodecyl sulfate (SDS) vs. Using Western blot methodology, we systematically examined: (1) gel composition (Tris-glycine vs. In order to assess collagen synthesis and extracellular accumulation in fibrotic disorders, improved methods are needed to detect changes in procollagen versus mature collagen at the protein level. Fibrillar collagen type 1 is the most abundant type of collagen within the body and is a critical component of extracellular infrastructure.