This method can help answer key questions about perturbations in signaling networks that can cause cancer growth or the development of resistance to specific therapies. The main advantage of this technique is that it can provide rapid and reliable feedback on the phosphorylation state of multiple networks that are frequently deregulated in cancer. The implication of this technique extends towards therapy of drug-resistant cancers because identification of alterations and post-translational modifications can elucidate changes in signal transduction pathway activity.
While this technique is used on phosphorylation, it can also be used to look at other post-translational modifications such as glycosylation and ubiquitylation. Begin by thoroughly rinsing overnight cultured cells three times with 10 milliliters of PBS taking care to remove all of the PBS after the last wash. Next, add the appropriate volume of lysis buffer from the protein assay kit and use a cell scraper to detach the cells.
Triturate the resulting cell solution approximately 10 times before transferring at least one times 10 to the seventh cells to a 1.5 milliliter tube. Incubate the cells for 30 minutes at four degrees Celsius with shaking. Then load the lysate into a syringe equipped with a 27 gauge needle and aspirate and dispense the sample 10 times to shear the cell membranes.
When the cell membranes have been completely disrupted, centrifuge the lysate and transfer the supernatant to a new 1.5 milliliter tube for subsequent total protein quantification according to standard protocols. To perform a human phosphokinase assay, first use flat-tipped tweezers to place one array membrane into each well of a four-well multi-tray containing two milliliters of freshly prepared array buffer one per well with the numbers of the membranes facing up. Upon submersion, the dye on the membrane will disappear.
Cover the tray with a lid and incubate the membranes on a rocking platform shaker for 60 minutes at room temperature. During this blocking incubation, dilute each lysate sample to a 50 to 100 micrograms of protein concentration in a maximum volume of 334 microliters of lysis buffer and bring the final volume of extracted protein sample up to 1.5 milliliters with fresh array buffer. Next, carefully aspirate the array buffer from each well of the multi-tray and incubate the membranes with 1.5 milliliters of protein sample per well overnight at two to eight degrees Celsius on a rocking platform shaker.
The next morning, carefully transfer each array into individual plastic containers containing 20 milliliters of wash buffer for three 10-minute washes on a rocking platform at room temperature. After the last wash, add 20 microliters of the appropriate reconstituted antibody cocktail to two milliliters of array buffer, two per well, and carefully blot the lower edge of each membrane on a paper towel to remove any excess wash buffer. Then place each array back into the appropriate well of the tray of antibody and cover the tray for a two-hour incubation at room temperature on a rocking platform.
At the end of the incubation, carefully transfer each array into a new eight by 11 centimeter plastic container containing 20 milliliters of wash buffer for three 10-minute washes as just demonstrated. After the last wash, add one milliliter of an appropriate streptavidin horse radish peroxidase solution to each well of the multi-well tray and return the membranes to their appropriate wells for a 30-minute incubation at room temperature. At the end of the incubation, place each membrane between two pieces of five millimeter filter paper to remove any excess buffer and incubate the dried membranes with an appropriate horse radish peroxidase detection solution for three minutes.
Then place the membranes into individual clear plastic sheet protectors for subsequent imaging. To assess the receptor tyrosine kinase expression in each sample, open the images in an appropriate image processing program such as Image J and select Edit and Invert from the appropriate dropdown menus to invert the black spots on the white background of the image to white spots on a black background. Next, use the Oval tool to encircle one pair of white dots on the dot blot and select Analyze and Measure to automatically open a new window displaying the values for the selected area.
Then place the point of the mouse arrow into the center of the oval and drag the circular area around the next dot area until all the spots of interest have been measured. In this representative experiment, the effects of tyrosine kinase inhibitor resistance on signal transduction pathways was analyzed in one control in three tyrosine kinase inhibitor resistant cell lines. Six phospho proteins with differential activity were chosen and the relative intensity was determined for several candidate proteins in each cell line.
As demonstrated by the heat map, a semi-quantitative readout on the changes in the protein phosphorylation of important signal transduction proteins could then be generated. While attempting this procedure, it's important to remember to start with healthy and actively dividing cells as stressors may induce changes in signal transduction pathway activity. Following this procedure, other methods such as digital PCR and immunoblotting can be used to answer additional questions such as do changes in phosphorylation lead to the activation of transcription factors?
After its development, this technique has paved the way for researchers in cancer biology to explore fundamental changes in the post-translational modification of proteins leading to the advent of personalized medicine for patients with cancer. Don't forget that working with cancer cell lines is extremely hazardous so be sure to use aseptic techniques, caution when disposing of sharps and wear a lab coat at all times.
