Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

The overall goal of this procedure is to quantify the relative changes in the level of a protein at Centrosomes under various conditions. For example, at a specific point in the cell cycle after inhibition of the proteasome, this is accomplished by first treating cells that are growing on cover slips with either the proteasome inhibitor MG one 15 or with the control solvent DMSO. Then immediately adding BRDU to both cells and to incubating them for four hours in a cell culture incubator.

The second step is to transfer the cover slips to a 24 well plate to fix the cells. Next, the cells are stained with antibodies against BRDU and the proteins of interest, and then with corresponding secondary antibodies. The final step is to acquire images of BRDU positive cells using a fluorescence microscope and digital imaging software to apply identical exposure time and Z axis length to cells from each sample.

Then analyzing the images to quantify the relative level of romal VDA C3.Ultimately, quantitative immunofluorescence microscopy is used to show that the Centro somal pool of VDA C3 is increased by twofold when the proteasome is inhibited during S-phase. The main advantage of this technique over existing methods like flow cytometry, immuno blotting, or quantitative microscope, is that this technique analyzes the relative romal level of the protein in two differently treated samples that are processed on two different cover slips. This method uses normalization to an internal standard to control for the need to process the two experimental cell populations.

Separately, Though this method can provide insight into the regulation of specific proteins at centrosomes. It can also be applied to other situations such as other subcellular structures or examining specific pools of proteins that are found at multiple sites within the cell. Demonstrating the procedure will be schu majumder, a postdoc from my laboratory To begin the experiment passage two times 10 to the fifth asynchronously growing RPE one cells into previously prepared 35 millimeter dishes with cover lips and grow the cells in two milliliters of complete medium.

Replace the culture medium every 24 hours with fresh prewarm medium After 44 hours. Replace the culture medium with complete medium containing 0.05%DMSO or MG one 15 at a final concentration of five micromolar, and simultaneously add BRDU to the cells at a final concentration of 40 micromolar incubate the cells for four hours. Next, transfer each cover slip to a 24 well plate.

Add 500 microliters of pre tilled methanol to each well and incubate the plate at minus 20 degrees Celsius to fix the cells. After 10 minutes, immediately wash the cover slips three times with 500 microliters of wash buffer. Incubate the fixed cells on cover, slips in 200 microliters of blocking buffer for 30 minutes while the cells are incubating.

Prepare a humidified chamber by placing a wet paper towel in the bottom half of an empty 1000 microliter pipette tip box. Lay a strip of parfum on the rack surface and add a 20 microliter droplet of the rabbit anti V DDA three and mouse anti gamma tubulin antibody diluted in blocking buffer onto the paraform. After blocking, invert a cover slip onto each droplet of antibody solution.

Ensure the cells are immersed and close the lid of the tip box. Incubate the cells with primary antibodies overnight at four degrees Celsius in the humidified chamber. Invert the cover slips again and return them to the 24.

Well dish wash the cover slips, then incubate them in a 150 microliter solution of secondary antibodies, diluted in blocking buffer for one hour at room temperature After incubation, wash the cover slips three times with wash buffer. Fix the stained RPE one cells with 500 microliters of methanol chilled at minus 20 degrees Celsius. After the cells have fixed for 10 minutes, wash them three times in wash buffer.

Next, incubate the cells in 200 microliters of two normal HCL for 30 minutes. At room temperature, neutralize the cells with 300 microliters of one molar tris CL pH eight, and wash the cells three times. With wash buffer block the cells with 200 microliters of blocking buffer for 30 minutes at room temperature.

After blocking, incubate the cells with rat anti BRDU antibody and blocking buffer for 45 minutes at 37 degrees Celsius in the humidified chamber. Return the cover slips back to the 24. Well dish and wash the cells.

Next, incubate them with secondary antibody diluted in 150 microliters of blocking buffer. In a 24 well dish for one hour at room temperature. Then wash the cover slips three times with one XPBS spot, a six microliter droplet of a mounting solution containing Antifa reagent on a glass microscope.

Slide invert a cover slip with the cell side facing down onto the mounting solution. With a soft cleaning tissue, gently press the cover slip against the slide to remove excess liquid seal the cover slip onto the microscope. Slide by applying nail polish along the edge of the cover slip.

Use a 100 x plan APO oil immersion objective with a 1.4 numerical aperture to acquire the images of BRDU positive RPE one cells at room temperature. Determine the appropriate top and bottom focal plane along the Z axis with the 0.2 micron step size, and acquire images along the Z axis. Next, perform deconvolution of all image stacks acquired along the Z axis.

Obtain the total intensity projection of each image. Stack along the Z axis for cells whose centrosomes are separated by less than two microns. Draw a small square of 20 to 30 pixels per side around both centrosomes and mark the selected area.

Draw a larger square of 24 to 35 pixels per side around the first square and mark the selected area of the large square. Then obtain the area and the total fluorescence intensity of each fluoro four. In each box, calculate the background corrected fluorescence intensity of each fluoro.Four.

Finally obtain the normalized fluorescence intensity of VDA C3 by calculating the ratio of the background corrected fluorescence intensity of its fluoro. Four to that of the fluoro four used for the chosen internal standard images were captured of random fields of asynchronously growing RPE one cells incubated with BRDU and either the proteasome inhibitor MG one 15 or the control solvent DMSO and display DNA and BRDU. The background corrected fluorescence intensity corresponding to Centro Somal.

VD three was roughly 2.5 fold higher in MG one 15 treated cells then in control cells as demonstrated by this box and whisker diagram. When the data was normalized for total VD three fluorescence against that of gamma tubulin, the fold increased, dropped slightly to roughly twofold. VD C3 staining at non romal cytes or the total cellular level of VD three was not affected by proteasome inhibition.

Thus, the romal pool of VDA C3 is regulated by proteasome mediated degradation. While attempting this procedure, it's important to remember that the antibodies against the test protein and the internal standard must be raised in different host species and that the images of both samples should be acquired using identical imaging parameters, such as the number of C-sections and exposure times in order to minimize the effect of digital imaging.