13.4: Exercise 2 - Preparing cell extracts
- Page ID
- 17576
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Prepare the cells for extraction
- Collect your team’s 6 cultures and note which cultures contain glucose and which cultures contain galactose.
- Determine the cell concentration or each culture. Vortex the tubes and transfer 100
μL of each cell culture to 900 μL deionized water. Measure the OD600 of cultures in the spectrophotometer. Note the values in your notebook. You will refer to them later when interpreting your SDS-PAGE gels (Chapter 14). - Transfer 1.5 mL of your cultures into labelled microcentrifuge tubes. Collect the cells by centrifugation for 1 minute using the microcentrifuge at top speed. Remove and discard the supernatant.
- Rinse the cells. Add 1 mL deionized water to each tube. Resuspend the cell pellets by gently drawing the cells in and out of a micropipette tip, taking care to prevent premature lysis of the cells. This rinse step removes proteins from the culture medium that may contaminate the cell pellet. Centrifuge again for 1 minute at top speed.
- Discard the supernatant and resuspend the cells in 100 μL deionized water.
Prepare the protein extract
- Add 100 μL of 0.2N NaOH to each tube, and incubate the cells for 5 minutes at room temperature. (The addition of NaOH does not lyse the cells, but it makes them more permeable and more fragile.)
- Pellet the cells again in the microcentrifuge. Remove and discard the supernatant. (The proteins that you are interested in are in the pellet.)
- Resuspend the cells in 50 μl 2 X SDS-PAGE sample buffer.* Ensure that the tubes are tightly capped. IMMEDIATELY place the tubes in a boiling water bath. Leave the cells in the water bath for 3 minutes. This treatment effectively denatures the proteins. Yeast cells contain many proteases that could degrade other cellular proteins, so it’s important that the proteases are denatured before they have the chance to attack other cellular proteins.
NOTE: The 2 X SDS-PAGE sample buffer contains 2-mercaptoethanol (also known asb-mercaptoethanol, or BME). Use appropriate caution and work quickly when handling this reagent. BME is a volatile reagent with a strong odor reminiscent of rotten fish. - After boiling, store the samples in the freezer for future use.
*2 X SDS-PAGE sample buffer consists of:
120 mM Tris/HCl, pH 6.8
10% glycerol (glycerol is significantly more dense than water) 4% SDS
8% 2-mercaptoethanol
0.004% bromophenol blue (a tracking dye for electrophoresis)