Phosphatase Assay in CRC Cells: A Method to Examine the Phosphorylation Status of Tau Protein in Colorectal Cancer Cells

- Tau protein localizes peripherally to the nucleolus in colorectal cancer cells and helps in maintaining nucleolar structure. Mostly, these cells express hyperphosphorylated tau protein, which results in drug resistance. Thus, we analyze the levels of tau phosphorylation by performing a phosphatase assay.

Begin by taking the cell lysate containing total protein from human colorectal cancer cells in two vials labeled as test and control. Treat the test sample with alkaline phosphatase enzyme. Alkaline phosphatase removes the phosphate groups from tau, whereas the control vial retains phosphorylated tau protein.

Next, run both the sample and control tau on a polyacrylamide gel to separate the proteins. Phosphorylated tau moves slower on the gel than non-phosphorylated tau protein. Electroblot the protein sample to a suitable membrane and add primary antibodies corresponding to both phosphorylated and normal tau.

Incubate the blot overnight. Treat the blot with reporter enzyme-conjugated secondary antibody that binds to the Fc region of primary antibody. Finally, develop the blot using chemiluminescent substrate to identify tau protein from the total protein sample present in the blo