The answer depends on the question: Optimal conditions for western blot characterization of muscle collagen type 1 depends on desired isoform

Abstract

Fibrillar collagen type 1 is the most abundant type of collagen within the body and is a critical component of extracellular infrastructure. 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. Using Western blot methodology, we systematically examined: (1) gel composition (Tris-glycine vs. bis-Tris, gradient vs. non-gradient, sodium dodecyl sulfate (SDS) vs. no SDS); (2) sample preparation (SDS vs. no SDS, β-mercaptoethanol (BME) vs. no BME, boiling vs. no boiling); and (3) running buffer composition (SDS vs. no SDS). Our results indicate full native gel conditions prevent resolution of all collagen type 1 bands. 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. Also, BME must not be added to the sample buffer and samples should not be boiled. 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. 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. Thus, the exact parameters employed are dependent upon the collagen protein product that the scientist desires to identify.

Blocking CTGF/CCN2 reduces established skeletal muscle fibrosis in a rat model of overuse injury

“…For CCN2, gels were made as described earlier for CCN1 and 3. For collagen type I, 8% gels were made without SDS in the gel, yet with SDS in sample and loading buffers, as described 38 . The antibodies used detected bands at the correct molecular weights (Figure S1A,B).…”

Forced treadmill running reduces systemic inflammation yet worsens upper limb discomfort in a rat model of work-related musculoskeletal disorders

“…Additionally, flexor digitorum muscles were collected from one arm of four of the FRC rats prior to perfusion. These samples were homogenized in phosphate buffered saline containing protease inhibitors, and total protein quantified, as previously described in detail [52]. These samples were prepared for gel electrophoresis by either: 1) boiling and exposing to beta-mercaptoethanol (BME), 2) not boiling the samples before exposing them to BME, 3) or neither boiling nor exposure to BME, in order to detect procollagen, mature collagen or cleavage products, as previously described in detail [52].…”

“…These samples were homogenized in phosphate buffered saline containing protease inhibitors, and total protein quantified, as previously described in detail [52]. These samples were prepared for gel electrophoresis by either: 1) boiling and exposing to beta-mercaptoethanol (BME), 2) not boiling the samples before exposing them to BME, 3) or neither boiling nor exposure to BME, in order to detect procollagen, mature collagen or cleavage products, as previously described in detail [52]. All samples were run on a 4-12% Tris-Glycine gel without SDS in the gel, yet with SDS in the sample and loading buffers [52].…”

“…These samples were prepared for gel electrophoresis by either: 1) boiling and exposing to beta-mercaptoethanol (BME), 2) not boiling the samples before exposing them to BME, 3) or neither boiling nor exposure to BME, in order to detect procollagen, mature collagen or cleavage products, as previously described in detail [52]. All samples were run on a 4-12% Tris-Glycine gel without SDS in the gel, yet with SDS in the sample and loading buffers [52]. After immunoblotting, blots were probed with an antibody against collagen type I (C2456, Sigma-Aldrich, St. Louis, MO).…”

Forced Treadmill Running Reduces Systemic Inflammation Yet Worsens Upper Limb Discomfort in a rat model of Work-Related Musculoskeletal Disorders

“…Additionally, flexor digitorum muscles were collected from one arm of four of the FRC rats prior to perfusion. Samples were homogenized in phosphate buffered saline containing protease inhibitors, and total protein quantified, as previously described in detail [52]. These samples were prepared for gel electrophoresis by either: 1) boiling and exposing to beta-mercaptoethanol (BME), 2) not boiling the samples before exposing them to BME, 3) or neither boiling nor exposure to BME, in order to detect procollagen, mature collagen or cleavage products, as previously described in detail [52].…”