— Ryla Cantergiani 2021/08/15 18:13

This protocol is for the preparation of protein extracts in yeast. Protein extracts may be used for proteomics analysis, such as with mass spectroscopy.

Protocol adapted from the Clontech Yeast Protocols Handbook (2009). https://www.takara.co.kr/file/manual/pdf/PT3024-1.pdf

Preparation of Yeast Culture

• YPD and appropriate SD medium liquid
• 15- and 50-mL conical tubes
• ice-cold diH2O
• Dry ice and ethanol, or liquid nitrogen
• Ice (~25 mL per sample)

Preparation of Protein Extracts

• 1.5-mL screw-cap microcentrifuge tubes (one per sample)
• glass beads
• Protease inhibitor solution
• 100X PMSF stock solution
• Cracking buffer stock solution
• Cracking buffer, complete

Cracking Buffer Stock Solution

Cracking Buffer (Complete)

The glass beads are stored with the RNase-free materials. It is important that these materials remain RNase-free. Please ask for assistance when collecting the glass beads. Here you cleanly list all of the reagents and equipment in our lab or elsewhere involved in performing this protocol, with links to our wiki reagent or equipment pages if necessary (make as needed the first time you enter a protocol- a protocol is not complete without completed links to all reagents and equipment). Please include this information in a table

Preparation of Yeast Culture for Protein Extraction

Day 0

1. For each yeast strain, prepare a 5-mL overnight culture in appropriate media (ex: YPD, YPD + drug, selection media).

Day 1

1. Before starting the protocol, cool the large tabletop centrifuge to 4°C.
2. Grow overnights until the OD600 reaches 0.4-0.6. Multiply the OD600 by the culture volume to obtain the total number of OD600 units. (You will need this number later).
3. Quickly chill the culture by pouring it into a pre-chilled 50-mL conical tube filled halfway with ice
4. Immediately place tube in the pre-chilled tabletop centrifuge and run at 1000 x g for 5 minutes at 4°C.
5. Pour off supernatant and resuspend the cell pellet in 50mL of ice-cold H2O. Any unmelted ice will pour off with the supernatant.
6. Recover the pellet by centrifugation at 1000 x g for 5 minutes at 4°C.
7. Resuspend in 1 mL H2O, and transfer to a labeled screw-top tube. Repeat centrifugation and remove supernatant.
8. Immediately freeze the cell pellet by placing the tubes in a dry ice/ethanol bath or using liquid nitrogen. Store cells in -80°C freezer until you are ready to proceed with the experiment.

Preparation of Protein Extracts Note: Unless otherwise stated, keep protein samples on ice.

1. Prepare complete cracking buffer and pre-warm it to 60°C. Because the PMSF degrades quickly, prepare only the amount of cracking buffer you will need immediately. Use 100 μL of cracking buffer per 7.5 OD600 units of cells.
2. Pre-heat the 95°C heat block to 100°C. Preheat incubator to 70°C. Cool centrifuge to 4°C.
3. Quickly thaw cell pellets by separately resuspending each one in the prewarmed cracking buffer. Because the initial excess PMSF in the cracking buffer degrades quickly, add an additional aliquot of the 100X PMSF stock solution to the samplse after 15 minutes and approximately every 7 minutes thereafter until Step 9 (CHECK NUMBER - freezing). Use 1 μL of 100X PMSF per 100 μL of cracking buffer.
4. Transfer each cell suspension to a 1.5-mL flip top tube containined 80 μL of glass beads per 7.5 OD600 units of cells. The volume of glass beads can be measured using a graduated 1.5-mL microcentrifuge tube.
5. Heat samples at 70°C for 10 minutes. Place tube tabs on samples before placing in the incubator.
6. Vortex vigorously for 1 minute.
7. Pellet debris and unbroken cells in a microcentrifuge at 14000 rpm for 5 minutes at 4°C.
8. Transfer supernatants to 1.5-mL screw-cap tubes and place on ice. This is the first supernatant.
9. Treat the pellets as follows:

• Place tubes in the 100°C heat block for 3-5 minutes.
• Vortex vigorously for 1 minute.
• Pellet debris and unbroken cells in a microcentrifuge at 14000 rpm for 5 minutes at 4°C.
• Add 50-100 μL of cracking buffer. Repeat centrifugation.
• Combine supernatant with first supernatant in screw-top tube.

1. Boil the samples briefly. Samples may be stored on dry ice or in a -80°C freezer. Run a Bradford assay to measure the approximate concentration of your protein extracts.