12/28/2023 0 Comments Licor western blot boxesRight blots show Ponceau red staining of membranes prior to antibody probing. Left blots were probed with anti-collagen type 1 antibodies. Hindlimb soleus muscle lysates were treated with or without boiling, and with or without BME, prior to being run on 8%, 6% or 4%–12% Tris-glycine gels with no SDS in the gels, running or sample buffers (2× native sample buffer used). Native and modified native western blot results for rat lysates on varying percent Tris-glycine gels. Further processing produces cleaved collagen I products that range between ~50–80 kDa in muscles. Collagen a1(I) and a2(I) from which the N- and C-terminals have been removed are secreted and function as the building block for collagen fibril formation. Cleavage of first the N-terminus propeptide and then the C-terminus propeptide yields mature collagen a1(I) and a2(I) (~100 to 140 kDa dependent on tissue type, preparation and assessment conditions). The observed molecular weight ranges of procollagen a1(I) and a2(I) chains are ~140–200 kDa. The intact disulfide bonded collagen type 1 has a molecular weight range of ~250 and higher under non-reduced conditions (no BME). As shown in Figure 1, collagen I precursors are complex proteins composed of three polypeptide chains called α chains (two α1 chains and one α2 chain) wound together in a right handed triple helix. Therefore, the focus of this protocol is on western blot characterization of collagen type 1 chain subtypes.Ĭollagen is known to be derived from a larger precursor that is subsequently modified via cleavage events to smaller derivatives, which can vary between tissues and during different physiological circumstances depending on the relative rates of biosynthesis and degradation. 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. 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.
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