Microtubule Binding Assay in CRC Cells: A Method to Examine Microtubule Binding Tau Protein in Colorectal Cancer Cells
Transcript
– 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 protocol, we will perform the microtubule binding assay to identify microtubule-bound proteins.
– After preparing fresh samples, incubate them at 37 degrees Celsius for 30 minutes. Ultracentrifuge the samples at 100,000 x g and 25 degrees Celsius for 60 minutes. Next, transfer 120 microliters of each supernatant containing unbound tau to separate clean and labeled tubes. Add 120 microliters of 5X sample buffer to the pellet which contains bound tau.
After mixing thoroughly by vortexing and pipetting, transfer the mixture to clean labeled tubes. After preparing samples of equivalent protein concentration, add fresh 4X SDS gel-loading buffer containing 400 millimolar DTT. Use a heat block to incubate the samples at 100 degrees Celsius for five minutes.
Vortex the samples, and then let them cool at room temperature for 10 to 15 minutes. Load 13 microliters of sample per well and the molecular weight ladder on a 10% polyacrylamide gel. Connect the positive and negative electrodes of the electrophoresis system to a power source, and then set the voltage to 70 volts for 20 minutes.
After this, increase to 125 volts and run the gel for approximately 70 to 120 minutes until the samples and protein ladder reach the end of the gel. Use the wet electroblotting system to transfer the gel onto a polyvinylidene fluoride membrane at 100 volts for about 100 minutes. Incubate the membrane to blot in 4% bovine serum albumin for 90 minutes at room temperature. Then, incubate overnight in primary antibody solution at 4 degrees Celsius.
The next day, wash the blot four times with PBST with each wash lasting seven minutes. Incubate the washed blot in a relevant secondary antibody solution for 90 minutes at room temperature. Then, wash with PBST for seven minutes repeating the wash a total of four times. Develop the blot using a chemiluminescence kit. Cover with transparent plastic wrap and acquire images using a chemiluminescence imaging system.
