1. Microtubule-binding Assay
- 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.
- 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.
- Prepare samples as per Table 1.
- Incubate at 37 °C for 30 min. Ultracentrifuge at 25 °C for 60 min at 100,000 x g.
- Transfer 120 µL of supernatants containing unbound tau, to clean and labeled tubes.
- 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.
- 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.
- 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.
- 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.
- Assemble an electrophoresis system for running a 10% polyacrylamide gel by SDS-PAGE.
- Load 10 µg protein (13 µL sample) per well on a 10% polyacrylamide gel. Load the molecular-weight ladder.
- 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.
- After completion of electrophoresis, transfer the gel onto a PVDF membrane using a wet electroblotting system. Transfer for ~100 min at 100 V.
- 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.
- Block the membrane in 4% BSA blocking buffer for 90 min at room temperature.
- 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.
- 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.
- 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.
| Sample | Name | Microtubule needed | Main Protein needed | PEM-GTP-PTX |
| 1 | HCT 116-Control (6.21 μg/μl) | 2 μl | 9.66 μl (60 μg) | 108.34 μl |
| 2 | HCT 116-Curcumin 10 μM (4.81 μg/μl) | 2 μl | 16.63 μl (80 μg ) | 101.37 μl |
| 3 | HCT 116-Curcumin 20 μM (3.28 μg/μl) | 2 μl | 30.49 μl (100 μg) | 87.51 μl |
| 4 | HCT 116-LiCl 25 mM (5.43 μg/μl) | 2 μl | 18.42 μl (100 μg) | 99.58 μl |
| 5 | E. coli tau | 2 μl | 1 μl tau-352 | 117 μl |
| 6 | MT Only | 2 μl | X | 118 μl |
| | | | 120 μl each |
