Yeast Cell Lysates

Modified from the Rine Lab

1. Grow cells to 0.3 to 0.5 O.D.(600) (~1E7 cells/ml). Use about 1.0 to 5.0 OD of cells per lysate; if your cells are at 0.5 OD per ml, use 2 ml to give you 1.0 OD of cells in your lysate. Use 2.0ml eppendorf tubes as the flat bottoms make glass bead lysis easier.
    
2. Harvest cells by spinning in micro-centrifuge for 2 minutes,decant supernatant.
    
3. Add 200 ul of SUMEB buffer + protease inhibitors.
    
4. Add 100 ul of 0.5 mm Acid Washed Glass Beads.
    
5. Vortex in multivortexer or by hand, 3 X 1 min speed setting 7 on our current machine.
    
6. Incubate for 10 min at 65°C or whatever temperature is desired.
    
7. Remove the lysate from the beads with a blue pipette tip to a new 1.5 ml eppendorf tube.
    
8. Spin 5 minutes to clarify. Remove supernatant to a new 1.5 ml eppendorf tube.
    
9. Use supernatant directly to load gel or dot blot.

Note that protease inhibitors are added from stocks to give 50 fold dilution.
Example: 20 ul of stock per 1ml buffer.
Use IMMEDIATELY after adding protease inhibitors.

 

SUMEB BUFFER (1% SDS, 8M Urea, 10mM MOPS, pH 6.8, 10mM EDTA, 0.01% bromophenol blue)

TO MAKE 100ml:

• 1.0ml 1M MOPS, pH6.8 stock solution
• 1.0g SDS, or 10ml 10% SDS stock solution
• 48.05g Urea
• 2.0ml 0.5M EDTA stock solution
• 1.0ml 1% bromophenol blue

This is used for lysing yeast at pH6.8. For pH8 lyses, use the variation of SUMEB with Tris buffer known as SUTEB.
Also, bromophenol blue can be left out if dye is not desired, such as using the lysate for immunopreicipations.

 

50X STOCK Protease Inhibitors (store at -20°C)
PMSF (87 mg/ml) ([500mM] phenylmethylsulfonyl fluoride)

TO MAKE 30 ml: Dissolve 2.61g PMSF in DMSO to equal a final volume of 30 ml.

LEUPEPTIN AND PEPSTATIN (5 mg/ml)

TO MAKE 10 ml: Dissolve 50mg LEUPEPTIN or PEPSTATIN in DMSO to equal a final volume of 10 ml.

TPCK (5 mg/ml) (tosylphenylalanine chloromethyl ketone)

TO MAKE 10 ml: Dissolve 50mg TPCK in DMSO to equal a final volume of 10 ml.
~undefined Note: The EDTA in the SUME buffer is also a protease inhibitor.

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Rapid Protein Prep

Horvath and Riezman, Yeast , 1994

Stuff you need:

Sample Buffer: 0.06M Tris-HCl, pH 6.8
10% (v/v) glycerol
2% (w/v) SDS
5% (v/v) 2-mercaptoethanol
0.0025% (w/v) bromophenol blue

1. Grow cells overnight (~1E7 cells/ml) and collect 1.5 ml cells in 1.5 ml microfuge tube (1 minute, 14000xg).
    
2. Wash cells 1X with water and collect again by centrifugation.
    
3. Resuspend cells in 100 µl sample buffer.
    
4. Heat at 95 deg C for 5 minutes.
    
5. Centrifuge 14000xg for 5 minutes.
    
6. Load ~25 µl per lane on an SDS polyacrylamide gel.
    

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Glass Bead Prep

Stuff you need:

Sample Buffer: 0.06M Tris-HCl, pH 6.8
10% (v/v) glycerol
2% (w/v) SDS
5% (v/v) 2-mercaptoethanol
0.0025% (w/v) bromophenol blue

0.1 M PMSF

0.5 M Benzamidine

1. Grow 25 ml of cells to mid-log.

2. Spin down (2500 rpm for 5 minutes). Wash 1X with water and spin again.

3. Resuspend in 1 ml of water and transfer to 1.5 ml microfuge tube. Spin down for 5 seconds and pour off water.

4. Resuspend in 0.5 ml of ice cold Sample Buffer with freshly added PMSF (0.5 mM) and benzamidine (0.5 mM).

5. Add glass beads (~0.5 ml).

6. Vortex on high 4X for 45 seconds with 30 seconds on ice in between each mixing.

7. Spin for 5 minutes in microfuge at 4 deg C.

8. Transfer supernatant to a new tube and boil for 5 minutes.

9. Load 10-15 µl on a protein gel.