Preparation of Yeast Protein Extracts

 

Preparation of Yeast Protein Extracts

 

 

(see Clontech YPH, p12)

 

 

 

Cracking buffer stock solution (see p54 Clontech YPH)

 

8M Urea; 5% w/v SDS; 40mM Tris-HCl pH6.8; 0.1mM EDTA; 0.4 mg/ml BMB in H 2 O

 

for a final volume of 20ml 9.6g Urea

 

1g SDS

 

0.8ml 1M TrisHCl pH6.8

 

4 µ l 0.5M EDTA

 

8mg Bromophenol Blue

 

 

 

Protocol

 

1. Set up 5ml O/N cultures in correct SD selection medium (SD-Leu or SD-Trp).

 

2. Also set up untransformed yeast as a control. Leave at 30 ° C, 230-250rpm, for 18-24h.

 

3. For each clone to be assayed, separately inoculate 50ml of YPDA with the entire O/N culture. Take an OD600 reading.

 

4. Incubate at 30 ° C, 230-250rpm, until the OD600 reaches 0.4-0.6 (this can take between 4-8h).

 

5. Use OD600 Units to determine the volume of each yeast culture to be taken; i.e. decide to take 15 OD600 Units of each culture 15 / OD600= volume to be taken, so if OD600=0.5, take 30ml

 

6. For each culture, transfer the volume calculated above into a 50ml Falcon half filled with ice. Spin at 2500rpm (MSE) for 5 min.

 

7. Pour off supernatant and resuspend pellet into 50ml of ice cold H 2 O and spin again.

 

8. Remove supernatant and freeze the pellet in liquid N 2 (cells can be stored at -80 ° C)

 

9. Just before use, make up complete cracking buffer