Biochemical Properties of CARM1: Impact on Western Blotting and Proteomic Studies.

Abstract

CARM1 is an arginine methyltransferase that has crucial roles in a number of cellular pathways and is being explored as a therapeutic target in diseases such as cancer and neurodegenerative disorders. Its deregulation at the protein level was found to have potential prognostic value, and as such, its protein levels are regularly assessed through the common practice of western blotting (WB). Our group uncovered that CARM1 has biochemical properties that complicate its analysis by standard WB sample preparation techniques. Here, we show that CARM1 has the ability to form SDS-resistant aggregates that effectively hinder gel migration in SDS-PAGE. CARM1 levels and the temperature at the denaturation step can both influence CARM1 aggregation, which prompts the use of additional measures to ensure representative detection at the protein level. We have demonstrated the formation of CARM1 aggregates in both cell and tissue extracts, making these findings an important consideration for any CARM1-related study. We also show how aggregate formation in models of CARM1 overexpression can hinder proteomic studies. Having identified factors that can induce CARM1 aggregation, we suggest alternative sample preparation techniques that allow for clear resolution of the protein in stringent denaturing conditions while avoiding aggregation.