This protocol measures subcellular protein ubiquitination levels and proteasome activity in the rodent brain allowing within subjects comparisons of how ubiquitin-proteasome activity changes in response to cellular activity, learning, or disease. The protocol allows collection of synaptic, cytoplasmic, and nuclear fractions from the same rodent brain. Minimizing loss, it could be performed with small tissue quantities and basic laboratory equipment.
Demonstrating the procedure will be Taylor McFadden, a graduate student from my laboratory. Begin this procedure with collection and dissection of rodent brain tissue as described in the text protocol. Ensure that the hemisphere used is counter balanced across extraction conditions for each experimental group.
Remove the 1.5 milliliter centrifuge microtube containing one hemisphere of the region of interest from the minus 80 degree Celsius freezer. Using a scalpel, transfer the frozen brain tissue to a two milliliter glass Teflon homogenizer. Add 500 microliters of lysis buffer to the Teflon tube.
Using pestle B, homogenize the same tissue with 15 strokes until no visible amount of solid material is present. Use a turning motion during each stroke. With a 1, 000 microliter pipette, transfer the homogenized sample to a new 1.5 milliliter microcentrifuge tube.
Place the tube on wet ice and incubate for 30 minutes. Place the tube in the microcentrifuge and spin for 10 minutes at 845 times g in four degrees Celsius. After completion, carefully remove the supernatant by pipetting and place into a new 1.5 milliliter microcentrifuge tube.
This is the cytoplasmic fraction. Add 50 microliters of extraction buffer to the resulting pellet and resuspend by pipetting. Do not vortex the pellet.
Place the tube containing the resuspended pellet on ice and incubate for 30 minutes. Then centrifuge the tube for 20 minutes at 21, 456 times g in four degrees Celsius. Following centrifugation, carefully remove the supernatant by pipetting and place into a new 1.5 milliliter microcentrifuge tube.
This is the nuclear fraction. Homogenize one hemisphere of the region of interest as before except to use 500 microliters of TEVP buffer instead of lysis buffer. Using a 1, 000 microliter pipette, transfer the homogenized sample to a new 1.5 milliliter microcentrifuge tube.
Centrifuge the sample at 1, 000 times g for 10 minutes at four degrees Celsius. Collect the supernatant and transfer it to a new 1.5 milliliter microcentrifuge tube using a 1, 000 microliter pipette. Centrifuge the sample at 10, 000 times g for 10 minutes at four degrees Celsius.
Discard the original pellet which contains nuclei and the large debris. Transfer the supernatant to a new 1.5 milliliter microcentrifuge tube. This is the cytosolic fraction.
Add 50 microliters of homogenization buffer to the pellet and resuspend by pipetting until no solid material is visible. Centrifuge the sample at 20, 000 times g for 10 minutes at four degrees Celsius. Transfer the supernatant to a new 1.5 milliliter microcentrifuge tube.
This is the crude synaptosomal membrane fraction. To set up the assay, prewarm the plate reader to 37 degrees Celsius and hold through the run. Set the excitation to 360 nanometers and emission to 460 nanometers.
If the 96 well plate used is clear, set the optics position to bottom. If a dark 96 well plate is used, set optics position to top. Program a kinetic run with a time of two hours scanning every 30 minutes.
Reconstitute the 20S 10X assay buffer provided in the kit with 13.5 milliliters of ultrapure water. Add 14 microliters of 100 millimolar ATP to the now 1X buffer. This significantly enhances proteasome activity in the samples and improves assay reliability.
Reconstitute the AMC standard provided in the kit with 100 microliters of DMSO. Perform steps using the AMC standard in the dark or under low light conditions as the standard is light sensitive. Create a standard curve of AMC from a series of high to low AMC concentrations.
This curve will be used for plate reader calibration and analysis of proteasome activity in the homogenized samples. Reconstitute the proteasome subtrate provided in the kit with 65 microliters of DMSO. Also perform this step in the dark or under low light conditions as the substrate is light sensitive.
Then create a one to 20 dilution of the proteasome substrate in a new 1.5 milliliter microcentrifuge tube using 20S assay buffer. Add a normalized amount of the desired samples to a 96 well plate. Run each sample in duplicate.
The amount of sample needed varies based on tissue preparation. Generally, 10 to 20 micrograms is sufficient for any subcellular fraction. Bring the sample well volume to 80 microliters with ultrapure water.
The amount added depends on the volume of sample added. In two separate wells, add 80 microliters of water alone. These will be the assay blanks.
Add 10 microliters of 20S assay buffer to each well including assay blanks. Use a repeater or automated pipette to ensure consistent assay volume across the wells. Turn off the lights or enter a dark room.
Add all 100 microliters of diluted AMC standards to a new well using a single well for each standard. In the dark, use a repeater or automated pipette to add 10 microliters of diluted proteasome substrate to wells containing sample and assay blanks but not the AMC standard. Place the plate into the plate reader and start the kinetic run.
Perform quantification of diverse polyubiquitin tags in different subcellular fractions collected from rodent brain tissue using a variety of standard Western blotting protocols in combination with unique linkage-specific polyubiquitin antibodies. Some ubiquitin Western blot images will provide columns of distinct bands while others produces smear-like pattern with few or no clear lines. For quantification of imaged ubiquitin Western blots, draw a box around the column that extends the entire molecular standards ladder.
Adjust the box if ubiquitin staining does extend through the entire ladder. This is common for lysine 48 modifications and varies widely across subcellular compartments. Finally, subtract out the background which is calculated as the mean optical density of the background immediately surrounding the column on all sides.
Shown here is the quantification of proteasome activity in different fractions collected from the lateral amygdala of the same animal. During the in vitro proteasome activity assay, relative fluorescent units detected increased from the beginning to the end of the assay in the synaptic fraction, the cytoplasmic fraction, and the nuclear fraction. The proteasome inhibitor beta-lac prevented RFUs from changing across the session.
Subcellular differences were observed in proteasome activity in the lateral amygdala of the same animal. An increase in nuclear proteasome activity was detected in trained animals relative to controls which corresponded to a decrease in activity within the synaptic fraction. Cytoplasmic proteasome activity remained at baseline.
Shown here are subcellular differences in linkage-specific protein ubiquitination in the lateral amygdala of the same animal following learning. There was an increase in overall ubiquitination in the nuclear fraction following learning which correlated with a decrease in the cytoplasmic fraction. Following learning, linear ubiquitination increased in the nuclear fraction but not cytoplasmic or synaptic fractions.
Interestingly, K63 ubiquitination increased in the nuclear fraction following learning which correlated with a decrease in the synaptic fraction. Whereas K48 uniquitination increased in the nuclear and cytoplasmic fractions following learning but not in the synaptic fraction. This protocol can also be used to understand the subcellular distribution and function of other proteins within the same animal.
