The overall goal of this indirect immunoflorescent assay is to optimize the localization and quantification of endogenous and exogenous centromere-kinetochore proteins, including centromere protein A and flag-tagged exogenous CENP-A proteins in human cells. This method can help answer key questions about how to identify and quantify centromere-kinetochore proteins. The main advantage of this technique is that it can be used to quanitfy different levels of centromere-kinetochore expression depending on the selected anaphase of interest.
18 hours after seeding, wash the cultured cells one time with PBS and add 500 microliters of reduced serum medium to each well of the six well polystyrene culture plate. Next, transfect the cells in each well with freshly prepared A and B mixture solution and incubate the cultures at 37 degrees Celsius and 5%carbon dioxide. After four and a half hours, change the medium to high glucose DMEM supplemented with FBS and antibiotics and return the plate to the cell culture incubator.
48 to 72 hours later, aspirate the cell culture medium and rinse the cells one time with PBS, adding the saline down the sides of the culture wells to avoid disturbing the cells. Then fix the cells with 4%paraformaldehyde for 30 minutes at 4 degrees Celsius. At the end of the fixation period, rinse the cells two times with Kb2 buffer and permeabilize the samples in Kb1 buffer for 30 minutes at room temperature.
Then rinse the cells one time with Kb2 buffer and block any non-specific binding with the addition of fresh Kb2 buffer. After five minutes at room temperature, use forceps to remove the seeded cover glasses from each of the wells, and use a hydrophobic barrier pen to draw hydrophobic barriers around each cover glass sample. Transfer the cover glasses into a new six well polystyrene plate and immerse the samples in the appropriate primary antibody for one hour at 37 degrees Celsius.
Then rinse the cells three times with Kb2 buffer and label them with the appropriate secondary antibody for another hour at 37 degrees Celsius. At the end of the incubation, rinse the samples with five 6 minute washes in Kb2 buffer. Then label the cell nuclei with Kb3 buffer containing DapE for five minutes at room temperature, followed by one to two washes in Kb2 buffer and refilling the wells with Kb2 buffer.
Now place a drop of mounting medium in the center of one microscope slide for each cover glass of cells, and remove any liquid from the cell samples. Finally, carefully place each sample onto the center of one of the prepared microscope slides, and remove any excess mounting medium with a paper towel. As observed in this representative experiment, the levels of endogenous CENP-A protein expression in the total cell lysates of CUL4A siRNA transfected cells were similar to the levels of CENP-A expression in Luciferase siRNA transfected cells.
Further, the protein levels of endogenous CENP-A in the total cell lysates of RBX1 siRNA transfected cells were similar to the endogenous CENP-A levels of the Luciferase siRNA transfected cells, suggesting the CUL4A RBX1 E3 ligase is required for the localization of CENP-A to the centromeres. CENP-A lysine mutants lose their ability to localize within the centromeres, suggesting that CENP-A K124 ubiquitylation is essential for the localization of CENP-A to the centromeres. Importantly, exogenous monoubiquitin fusion of CENP-A protein rescued the delocalization CENP-A K124R mutant, returning the protein to its centromeric location.
Once mastered, the cell fixation and immunoflourescent staining procedures can be completed in five to six hours if they are performed properly. Remember to gently apply all of the solutions onto the sides of the wells so that the cells won't be dislodged from their cover glasses when rinsing or immersing the samples. Don't forget that working with heated paraformaldehyde outside a ventilated room or that flammable materials near fire can be extremely hazardous and that the appropriate precautions should always be taken while performing this procedure.
