Methods to Study CCT Expression and Impact on Cytoskeleton

Chaperones are proteins whose function is to intervene in the correct folding of newly synthesized polypeptides in the ribosome, thus providing their native conformation. CCT belongs to the Hsp60 family of chaperonins and is specifically a Type II subgroup. Here, we describe methods for determining and manipulating the CCT protein and RNA levels, as well as CCT distribution in cells, using complementary biochemical and imaging approaches. Protocols are provided for samples from primary human cells and established cell lines, including electrophoretic separation and immunoblotting. In parallel, procedures for immunofluorescence staining and microscopy are outlined, with emphasis on fixation, permeabilization, and antibody optimization. Critical parameters, appropriate controls, and troubleshooting considerations are discussed to facilitate robust and reproducible detection of CCT across different cellular systems.