Microtubule Binding Assay in CRC Cells: A Method to Examine Microtubule Binding Tau Protein in Colorectal Cancer Cells

- Tau is a microtubule-associated protein involved in stabilizing and assembling the microtubules. Many cancer cells express hyperphosphorylated tau protein, which fails to maintain a well-organized microtubule binding. To study the interaction between tau protein and microtubules, add cancer cell lysate containing tau protein and a microtubule working solution in a microfuge tube.

The nonphosphorylated tau proteins present in the cell lysate bind to the microtubules. Now, add the desired amount of PEM buffer in the tube to stabilize the activity of tubulin, a protein that polymerizes into filaments to form microtubules. Incubate the mixture for the desired duration.

Centrifuge the mixture to pellet the microtubule-bound protein. The unbound protein remains in the supernatant. Transfer the supernatant to a fresh tube, and re-suspend the pellet in a suitable buffer. Add a gel loading buffer to the re-suspended pellet suspension and run the samples on a polyacrylamide gel to separate microtubule-bound protein and electroblot on a suitable membrane.

Add suitable antibodies and develop the blot using chemiluminescence substrate to identify the bound tau proteins. In the following proto