Methods for Measuring Type I Collagen Synthesis In Vitro
The excess accumulation of type I collagen within tissues leads to organ dysfunction and occurs as a result of an imbalance between synthesis and degradation. This chapter outlines several methods to assess the in vitro production of type I collagen that are employed in our laboratory. We describe Western immunoblotting of intact α1(I) collagen using antibodies directed to α1(I) collagen amino and carboxyl propeptides. The measurement of α1(I) collagen mRNA levels using real-time polymerase chain reaction is then outlined. Finally, methods to determine the transcriptional regulation of α1(I) collagen using a nuclear run-on assay are described.
Key WordsType I collagen Western immunoblot real-time PCR nuclear run-on assay
This work was supported by the National Institutes of Health grants K08-HL04232 and R01-HL66547 and the VA Research Enhancement Award Program.
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