Yeast Lac Z  Assay

b-Galactosidase Liquid Assay for Yeast

Modified From Ira Herskowitz’s lab  University of California, San Francisco

 Procedure

1. Grow the desired yeast strain to log phase in a liquid culture aim for OD600 less than 1.0. (0.7-0.8 is ideal).

 

2. Measure the Optical Density at 600 nanometers (OD600) by diluting 0.5 ml of culture in 0.5 ml of media (see Note #1).

 

3. Place 1 ml of culture into a microcentrifuge tube (see Note #2).

 

4. Microcentrifuge at maximum speed for 2 min.

 

5. Remove the supernatant, take care not to suck up the yeast pellet.

 

6. Add 1 ml of Z Buffer and mix well to resuspend the pellet.

 

7. Microcentrifuge at maximum speed for 5 min.

 

8. Remove the supernatant.  (Steps 9-12 can be replace by Y-Per lysis see Note #5)

 

9. Resuspend the cell pellet in 150 ul of Z Buffer with 2ME.

 

10. Add 50 ul of Chloroform (CAUTION! see Note #4).

 

11. Add 20 ul of 0.1% SDS.

 

12. Vortex the sample vigorously for 15 sec.

 

13. Add 700 ul of pre-warmed (30°C) ONPG Solution. Start timer at this point.

 

14. Incubate at 30°C while timing the reaction; remove aliquots at the appropriate time points (when yellow color develops see Note #3.

 

15. Stop reaction by adding 500 ul of 1 M Na2CO3 mix/vortex for 15 sec.

 

16. Microcentrifuge at maximum speed for 2 min. Transfer supernatant to a cuvette.

 

17. Determine the absorbance of the supernatant at 420 nm. The blank should be 700 ul ONPG solution +500 ul Stop solution. If using Y-Per reagent use this in the blank (see Note #5)

 

18. Calculate as follows:

 

Miller Units = (Å420*1000)/(Å600*min*ml)

 

Where:

 

Å420 is the absorbance units at 420 nanometers

 

Å600 is the absorbance units at 600 nanometers

 

min is the reaction time in minutes

 

ml is the reaction volume in ml

 

Solutions

Z Buffer                     

0.75 g KCl

Bring the final volume to 1 liter using ddH2O

16.1 g Na2HPO4, 7H2O

246 mg MgSO4, 7H2O

5.5 g NaH2PO4

 

1 M Na2CO3

Sodium Carbonate MW 105.99 g/mol

Therefore add 10.6g/100 ml of dH2O

 

ONPG                        

1 mg/ml ONPG (o-Nitrophenyl Β-D-Galactopyranoside)

Prepared in Z Buffer with 2ME

 

0.1% SDS

0.1% (w/v) SDS

 

Z Buffer with 2ME

Prepared in Z Buffer

Add 270 ul of 2-Mercaptoethanol (2ME) per 100 ml of Z Buffer

 

BioReagents and Chemicals

Magnesium Sulfate, Heptahydrate

Chloroform

Potassium Chloride

Sodium Carbonate

2-Mercaptoethanol (2ME)

ONPG

SDS

Sodium Phosphate Monobasic

Sodium Phosphate Dibasic Heptahydrate

 

Protocol Notes

 

1. When reading many samples, use a P200 pipette to remix the cells before reading (since the cells will settle to the bottom of the tube over time). Always determine the optical density of several identical samples and determine the mean optical density value.

 

2. Do each sample in triplicate, at least.

 

3. Until a color change is observed. Note: Hydrolysis of ONPG will lead to the production of a yellow color typically within minutes, but this is dependent on the amount of b-galactosidase present in the sample. Usually will take 20 min to 3 hour total reaction time.

Note: The reaction time will vary depending on the level of b-galactosidase expression in the test colony. To be within the linear range of the assay, the A420 should be between 0.02-1.0.

 

4. Chloroform is a biohazard use in the fumehood. Please consult this agent's MSDS for proper handling instructions.

 

 

5. Ian’s Modification using Y-PER

Also see Pierce Yeast b-Galactosidase Assay Kit manual

 

To bust open the yeast cells you can use Y-Per (Pierce) instead.

Follow the above procedure up to step 8.  Steps 9-12 are replaced by:

 

Pipet 250 ul of Y-PER™ Reagent into a microcentrifuge. Prepare one tube for each ml of culture to be assayed. Mix gently with a vortex mixer to create a homogenous solution 1-3 min. If the yeasts do not appear to by lysed then add another 100 ul of Y-PER or you may have to reduce the amount of yeast in step 2.

Goto step 13.

 

Note for blank:

250 ul of Y-PER™ Reagent, 700 ul of the ONPG, and 500 ul of the b-Galactosidase Assay Stop Solution (1M Na2CO3).