The overall goal of this procedure is to study tau phosphorylation, the microtubule binding capabilities of tau, and localization of tau, using confocal microscopy in colorectal cancer cells. This method can be effective for most of the biological research fields, especially cancer and dementia research, where phosphorylation and microtubule-binding are important factors. The main advantage of the presented phosphoratase assay is that it provides an easy way to perceive the whole phosphorylation of protein samples.
The main advantage is microtubule-binding assay is that, it is a simple procedure to separate the microtubule-binding proteins from nonbinding fractions. However, the main benefits of tau localization technique is that it requires the relatively small amount of primary and secondary antibodies. To begin this procedure, prepare all reagents as outlined in the text protocol.
Transfer approximately one million HCT 116 cells into culture dishes containing minimum essential medium supplemented with 10%fetal bovine serum and 1%penicillin streptomycin. When the cells reach 65%confluence, use an aspirator to remove the FBS supplemented medium. Then, add serum-free MEM, containing either curcumin or lithium chloride.
Incubate at 37 degrees Celsius in a humidified atmosphere containing 5%carbon dioxide for 24 hours. After this, wash the cells with 10 milliliters of iced cold PBS. Add one milliliter of ice cold PBS, and then use a cell scraper to scrape the cells and transfer them into 1.5 milliliter centrifuge tubes.
Centrifuge for four minutes at 1800 times G and four degrees Celsius. We suspend the cell pellets in 100 microliters of complete RIPA buffer at four degrees Celsius for 20 minutes, making sure to tap the tubes regularly to lyse the cells. Then, briefly sonicate the samples at an amplitude of 10%pulsing on for 0.2 seconds, and off for 0.2 seconds, for a total of 2 seconds.
Centrifuge the cell homogenate at 22, 570 times G and four degrees Celsius for 20 minutes. Transfer the supernatants into new labeled tubes and then measure the protein concentration by the Bradford assay, as outlined in the text protocol. 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 4XSDS 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 weighed 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 unsure 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.
After preparing fresh samples, incubate them at 37 degrees Celsius for 30 minutes. Ultracentrifuge the samples at 100, 000 times 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 Fibic 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 4XSDS 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 a 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 four degrees Celsius. The next day, wash the blot four times with PBST with each wash lasting seven minutes.
Incubate the washed blot in the 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. To begin, place cover slips into the wells of a six-well tissue cover plate, and then seed 250, 000 cells into each well in the recommended culture medium. After 24 hours, rinse the cells twice with PBS.
Add serum-free MEM, containing curcumin and incubate at 37 degrees Celsius in a humidified atmosphere containing 5%carbon dioxide for 24 hours. After this, wash the cells with ice cold PBS again. Fix the cells in 3.7%formaldehyde in a 37 degree Celsius incubator, in the dark.
Wash the cells with PBS, and then fix the cells in ice cold methanol at 20 degrees Celsius for 15 minutes. After this, incubate the cells on ice in 3%BSA and 0.1%Triconex 100 in PBS for 10 minutes. Wash the cells with PBS and then incubate in blocking buffer for one hour at room temperature.
Next, add 60 microliters of primary antibody solution to the cut polyvinylidene fluoride piece in each well. Placing the cover slips such that the cells make contact with the antibody solution. Incubate overnight at 4 degrees Celsius in a dark humid chamber.
The next day, wash the cells four times with PBS. Add 80 microliters of the secondary antibody solution to each of the polyvinylidene fluoride pieces, placing the cover slips such that the cells make contact with the antibody solution. Incubate in a dark humid chamber at room temperature for two hours.
After this, wash the cells four times with PBS. Mount the samples in the mounting medium with DAPI, and then fix the cover slips on glass slides. Then incubate in a dark chamber at room temperature for one and a half hours.
Using a confocal microscope examine the slides. In this study, cite specific tau phosphorylation is assessed using a phospho-tau antibody. Cells treated with curcumin are seen to express decreased tau as the concentration of curcumin increases.
Phospho-tau expression, on the other hand, is seen to increase at lower concentrations of curcumin, decreasing only at very high concentrations. Cells treated with lithium chloride are then examined. As can be seen, the expression of both tau and phospho-tau is significantly decreased under lithium chloride treatment.
Overall phosphorylation is then evaluated by phosphotase assay. Tau is seen to migrate faster in phosphotase treated samples compared with the untreated control sample, indicating that the control sample is phosphorylated. Curcumin treated cell samples showed very similar results, indicating that the treatment colorectal cancer cell lines did not reduce tau phosphorylation.
Both phosphatase treated and untreated samples electrophoresed nearly the same range in samples of cells treated with lithium chloride indicating a reduction in tau phosphorylation. Next, the microtubular binding assay of cell samples is successfully established using tau 352 as the positive control. As shown here, both curcumin and low concentration of lithium chloride treatment results in inhibited microtubule binding activity.
Confocal microscopy is then performed. Following curcumin treatment, tau is seen to translocate to the nucleus. This supports earlier studies, reporting that nuclear tau is key player in neuronal DNA protection.
After seeing this video, you should have a good understanding of how to perform a phosphotase assay to determine the whole phosphorylation of protein samples and how to perform a microtubule binding assay. You should be able to figure out how to execute an immunofluorescence assay by confocal microscopy using various smaller amount of antibodies. The procedures presented here are cost effective and will potentially help aid in the discovery and development of new theraputic agents to treat different tau-phorese and cancers.
