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

1. Phosphatase Assay

1. Treat the cell lysates when cells reach ~65% confluence (approximated using microscopy), remove the MEM supplemented medium and add serum-free MEM with 5 µM, 10 µM, and 30 µM curcumin or 25 mM, 50 mM, and 100 mM LiCl. Incubate at 37 °C for 24 h in a humidified atmosphere containing 5% CO₂, 24 h after treatment, wash the cells with 1 mL ice-cold phosphate-buffered saline (PBS), and scrape the cells using a cell scraper. Transfer the scraped cells into 1.5 mL centrifuge tubes and centrifuge for 4 min at 1,800 x g at 4 °C to obtain cell pellets. Lyse the cells by suspending the pellets in 100 µL of complete RIPA buffer at 4 °C for 20 min while regularly tapping the tubes. Sonicate the samples briefly. Centrifuge the cell homogenates in a benchtop centrifuge at 22,570 x g for 20 min at 4 °C. Transfer the supernatants to other labeled tubes using a pipette) with alkaline phosphatase in the alkaline phosphatase buffer.
   1. For both control and treated samples, prepare 20 µL samples containing 20 µg total protein. Add the volume of cell lysates that contains 20 µg total protein, followed by 2 µL alkaline phosphatase buffer and 10 µL alkaline phosphatase. Add distilled water to make up 20 µL.
2. Incubate the samples at 37 °C for 1 h. Stop the reaction by adding ethylenediaminetetraacetic acid (EDTA) at a final concentration of 50 mM, or by adding sodium orthovanadate (Na₃VO₄) at a final concentration of 10 mM. The reaction can also be stopped by heating at 75 °C for 5 min.
3. Before the incubation of samples with phosphatase finishes, prepare samples of the same concentration without adding phosphatase for comparison with phosphatase-treated samples.
4. Add the freshly prepared 4X SDS gel-loading buffer containing 400 mM DTT.
5. 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.
6. 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.
7. Block the membrane in a 4% BSA blocking buffer for 90 min at room temperature.
8. 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.
9. 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.
10. 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.