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

1. Microtubule-binding Assay

1. For the microtubule-binding assay, Add ~1 x 10⁶ HCT 116 cells into 100 mm tissue-culture dishes containing the Minimum Essential Media (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. After one day of culturing, cells will reach 60-70% confluence.
2. Prepare 1X (2 mL) PEM buffer from 10X PEM buffer stored at 4 °C by adding 20 µM (40 µL) paclitaxel and 1 mM (20 µL) GTP. Take the microtubule (MT) stock from -80 °C freezer and the E. coli tau working stock from -20 °C freezer.
3. Prepare samples as per Table 1.
4. Incubate at 37 °C for 30 min. Ultracentrifuge at 25 °C for 60 min at 100,000 x g.
5. Transfer 120 µL of supernatants containing unbound tau, to clean and labeled tubes.
6. Add 120 µL (same volume as supernatant) of 5X sample buffer to the pellet containing bound tau and mix thoroughly. Transfer the solution to clean labeled tubes and mix thoroughly.
7. Measure the protein concentration by the Bradford assay using commercial protein-assay kits.
   NOTE: While preparing samples, use the volume of solution (step 1.6) for preparing both the pellet and supernatant samples.
8. Prepare samples of equivalent protein concentration and add freshly prepared 4x SDS gel-loading buffer containing DTT (400 mM).
   NOTE: Samples can be stored at -20 °C at this stage.
9. Incubate the samples on a heat block at 100 °C for 5 min. Mix the samples using a vortex. Cool at room temperature for ~10-15 min.
10. Assemble an electrophoresis system for running a 10% polyacrylamide gel by SDS-PAGE.
    1. Load 10 µg protein (13 µL sample) per well on a 10% polyacrylamide gel. Load the molecular-weight ladder.
    2. After loading the sample, connect the positive and negative electrodes of the electrophoresis system to a power source to maintain a constant voltage. Initially, start at 70 V for 20 min to move the protein samples from the stacking gel into the resolving gel. Then, increase the voltage to 125 V and run the gel for approximately 70-120 min until samples and protein ladder reach the end of the gel.
    3. After completion of electrophoresis, transfer the gel onto a PVDF membrane using a wet electroblotting system. Transfer for ~100 min at 100 V.
    4. Mark the blot by making a small cut on the side of the molecular-weight ladder. Use a transparent plastic sheet and a sharp cutter for cutting the blot.
11. Block the membrane in 4% BSA blocking buffer for 90 min at room temperature.
12. Incubate the blot in primary-antibody solution (anti-tau at 1:5,000) at 4 °C overnight. Wash the blot in PBST four times, for 7 min each.
13. Prepare the relevant secondary-antibody solution (goat anti-mouse IgG at 1:5,000), and incubate the blot for 90 min at room temperature. Wash in PBST four times, for 7 min each.
14. Develop the blot using a chemiluminescence kit according to the manufacturer's recommendations. Cover the blot with transparent plastic wrap. Acquire images by a chemiluminescence imaging system for chemiluminescence with relevant software.

Table 1: Sample preparation for microtubule-binding assay.