Phosphatase Assay in CRC Cells: A Method to Examine the Phosphorylation Status of Tau Protein in Colorectal Cancer Cells
Transcript
– Tau protein localizes peripherally to the nucleolus in colorectal cancer cells and helps in maintaining nucleolar structure. Mostly, these cells express hyperphosphorylated tau protein, which results in drug resistance. Thus, we analyze the levels of tau phosphorylation by performing a phosphatase assay.
Begin by taking the cell lysate containing total protein from human colorectal cancer cells in two vials labeled as test and control. Treat the test sample with alkaline phosphatase enzyme. Alkaline phosphatase removes the phosphate groups from tau, whereas the control vial retains phosphorylated tau protein.
Next, run both the sample and control tau on a polyacrylamide gel to separate the proteins. Phosphorylated tau moves slower on the gel than non-phosphorylated tau protein. Electroblot the protein sample to a suitable membrane and add primary antibodies corresponding to both phosphorylated and normal tau.
Incubate the blot overnight. Treat the blot with reporter enzyme-conjugated secondary antibody that binds to the Fc region of primary antibody. Finally, develop the blot using chemiluminescent substrate to identify tau protein from the total protein sample present in the blot. In the following protocol, we will demonstrate the phosphatase assay to assess the overall phosphorylation status of tau in CRC cells.
– After preparing samples containing 20 micrograms of total protein, add two microliters of alkaline phosphatase buffer and 10 microliters of alkaline phosphatase to each. Then, add distilled water such that the final volume of each sample is 20 microliters.
Incubate the samples at 37 degrees Celsius for one hour. While the samples incubate, prepare additional samples at the same concentration, but without phosphatase for comparison. After one hour, stop the reaction by adding either EDTA at a final concentration of 50 millimolar, or sodium orthovanadate at a final concentration of 10 millimolar.
Add freshly prepared 4X SDS gel loading buffer containing 400 millimolar DTT. Using a heat block, 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.
Next, load 13 microliters of sample per well as well as the molecular weight ladder on a 10% polyacrylamide gel. Connect the electrophoresis system to a power source and set the voltage to 70 volts for 20 minutes to move the protein samples from the stacking gel into the resolving gel.
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. Using a wet electroblotting system, transfer the gel onto a polyvinylidene fluoride membrane at 100 volts for about 100 minutes.
Next, incubate the membrane to blot in 4% bovine serum albumin for 90 minutes at room temperature. Incubate the blot overnight in primary antibody solution at 4 degrees Celsius. The next day, wash the blot three times in PBST, washing for seven minutes each time.
Incubate in the relevant secondary antibody solution for 90 minutes at room temperature. Then, wash with PBST four times, with each wash lasting seven minutes. Develop the blot using a chemiluminescence kit according to the manufacturer's instructions.
Cover the developed blot with transparent plastic wrap and then acquire chemiluminescence images of the blot with a commercially available chemiluminescence imaging system.
