The overall goal of this experiment is to detect changes in protein insolubility with age in C.elegans and to evaluate how targeted gene knockdown influences this aging marker. This method can help answer key questions in the field of aging and proteostasis such as why organisms fail to maintain a healthy proteome with age. The main advantage of this technique is the opportunity to study protein aggregation during normal aging in the absence of disease processes.
Though this method can provide insight into protein aggregation in C.elegans, it can also be adapted to other model organisms, for example to study age-related protein insolubility in mouse. To begin treatment, add 207.45 milliliters of S Basal media with additional reagents as described in the accompanying text protocol to a 2, 800 milliliter Fernbach culture flask. Add a final concentration of 50 micrograms per milliliter of Carbenicillin and one millimolar of IPTG and close the flask with a membrane screw cap.
Take the L1's out of the 25 degree Celsius incubator and transfer them to 15 milliliter tubes. Centrifuge the L1's at 1, 900 times g for three minutes. After spinning, remove the supernatant.
Under a microscope, count the L1's per two microliters and average the numbers obtained from at least nine drops. Next, add 50, 000 worms for the young worm collection and 100, 000 worms for the aged worm collection into four Fernbach culture flasks prepared in the previous step. Then, add control RNAi bacteria and RNAi bacteria for the gene of interest proportionally to the number of worms.
After adding bacteria, complete worm culture with S Basal to bring the total volume to 300 milliliters. Incubate the worm culture at 25 degrees Celsius in a shaking incubator with 150 rpm until collection. Collect young worms at day two or three to measure basal levels of protein solubility.
Pour the worms from one flask into a separation funnel and let the worms sediment for 10 minutes at room temperature. After the worms sediment, open the stop cock and drip the worms into one 50 milliliter tube. Next, split the worm pellet into two 15 milliliter tubes.
Fill up the tubes to 15 milliliters with M9 and centrifuge the worms. To wash the worms, remove the supernatant and fill the tube up to 15 milliliters with M9.Repeat the centrifugation. Once washing is complete, transfer the worms to two 50 milliliter tubes and use ice cold M9 to fill up the total volume to 20 milliliters.
To remove bacteria and dead worms, add the two 20 milliliter diluted worm pellets to two 50 milliliter tubes filled with 20 milliliters of ice cold 60%sucrose. Quickly centrifuge the tubes. With a 25 milliliter pipette, carefully remove up to 15 milliliters of the top worm layer.
Place the top worm layer directly into 37 milliliters of M9 plus octoxynol-9 prepared on ice. Then, centrifuge the tubes at 2, 700 times g for three minutes at four degrees Celsius acceleration nine and deceleration seven. After discarding the supernatant, transfer the pellet into four 15 milliliter tubes and wash them twice with ice cold M9 plus octoxynol-9.
Then, centrifuge the tubes. Remove the supernatant and fill the tubes up to the 15 milliliter mark with ice cold M9 plus octoxynol-9. Wash the worms with ice cold M9 and combine the four tubes into two tubes.
Fill up the tubes to 15 milliliters and centrifuge. Remove the supernatant, fill up the two tubes with M9 at room temperature to a total volume of four milliliters and rotate them on a nutating mixer at 25 degrees Celsius for 40 minutes. After nutating, wash the worms twice with ice cold M9 plus octoxynol-9.
Then, wash them twice with M9 and transfer the worms to one tube. Wash the worms in the reassembly or RAB high salt extraction buffer without inhibitors before collection. Remove the supernatant until there is no liquid on top of the worm pellet.
Then, estimate the volume of the worm pellet and add an identical volume of RAB with inhibitors. Prepare a 50 milliliter tube half filled with liquid nitrogen on dry ice and using a Pasteur pipette, draw up the worms and slowly drip them into the tube. Allow the liquid nitrogen to evaporate and store the frozen worms at minus 80 degrees Celsius until further processing.
Cool down the mortar with liquid nitrogen and perform the animal disruption on dry ice. Transfer the frozen worms to the pre-cooled mortar and grind them for 2.5 minutes. Add 100 milliliters of liquid nitrogen to the powder and grind for another 2.5 minutes.
Use a microscope to check that the worm bodies are broken into small pieces. Then, transfer the powder into two milliliter tubes and store them at minus 80 degrees Celsius. On dry ice, weigh out two times 350 milligrams of ground worms per time point and per RNAi bacteria into two milliliter tubes.
To remove the high salt soluble proteins, add two volumes per weight of already prepared RAB with inhibitors, one millimolar PMSF, 200 units per milliliter DNase I, and 100 micrograms per milliliter RNase A to each tube and solubilize the powder on ice. Draw up the suspension into a one milliliter syringe 15 times and incubate it on ice for 10 minutes. Centrifuge at 18, 400 times g for 20 minutes at four degrees Celsius.
Go through the fat layer and collect the supernatant containing high salt soluble proteins into one two milliliter tube per condition. Remove the fat and discard it. Aliquot high salt soluble proteins to freeze at minus 80 degrees Celsius.
To discard lipids, solubilize the pellet with 700 microliters of RAB with inhibitors containing one molar sucrose without DNase I and RNase A.Draw up the suspension into a syringe 10 times and incubate it on ice for five minutes. Be sure to remove all of the supernatant and lipids and discard them. To remove SDS-soluble proteins, solubilize the pellet with 700 microliters of RIPA buffer.
Draw up the suspension into a syringe 10 times and incubate it for 10 minutes on ice. Centrifuge at 18, 400 g for 20 minutes at four degrees Celsius. Collect the supernatant containing SDS-soluble proteins and aliquot for freezing at minus 80 degrees Celsius.
Pool two samples of each condition together after solubilizing each pellet with 500 microliters of RIPA buffer and draw up the suspension into a syringe 10 times. Be careful to remove all of the supernatant and discard it. Solubilize the final pellet containing highly insoluble proteins with 400 microliters 70%formic acid and draw up the suspension into a syringe 20 times.
Incubate the suspension for 20 minutes on ice. Centrifuge the suspension in ultracentrifuge tubes to remove worm cuticle debris. Collect the supernatant that contains highly insoluble proteins and freeze it at minus 80 degrees Celsius.
Total protein staining of insoluble protein content of young and aged worms revealed a strong age-related increase in highly insoluble protein levels in control animals and not in long lived animals. Antibody staining of PAB-1, an RNA binding protein with a predicted prion-like domain, confirmed mass spectrometry data that long lived animals maintained PAB-1 soluble with age. In vivo analysis was performed of transgenic animals expressing tagRFP PAB-1 in the pharyngeal muscles.
In young animals, tagRFP PAB-1 was mainly diffusely located throughout the pharynx. With age, a progressive accumulation in bright puncta starting in the posterior pharyngeal bulb. During aging, we also observed aggregation in the anterior pharyngeal bulb in an increasing number of animals.
Quantification of different tagRFP PAB-1 aggregation levels with age in wild type and daf-2 mutant background reveals strongly delayed tagRFP PAB-1 aggregation with age in long lived animals. After watching this video, you should have a good understanding of how to collect a large number of worms for protein extraction and how to isolate highly insoluble large aggregates for further analysis by mass spectrometry or SDS page.
