Because of the simplicity and sensitivity of the β-galactosidase assay, yeast genes are frequently labeled with the functional portion of the lacZ gene to indicate the regulatory function of the expression of the yeast gene to be studied.

Operation method

β-Galactofuranosidase assay

Materials and Instruments

Yeast
YPD SDS Potassium Phosphate Na2CO3 OPNG
Water Bath Incubator Spectrophotometer Vortex Mixer Centrifuge

Move

1. Inoculate each single yeast colony with YPD (or appropriate) medium, pick 2~3 yeast containing lacZ fusion protein expression plasmid and incubate overnight at 30°C. If the fusion protein is on a plasmid, then incubate in the medium selected for that plasmid.2. Inoculate 20~50 μl of overnight culture in 5 ml of medium (or with appropriate selection medium and induction conditions). Cultivate cells to mid or late logarithmic growth: Enriched medium, 0. ^5~1×106 cells/ml or limiting medium, ^2~5×107 cells/ml ( _OD600=0.5~1.0).3. Collect cells by centrifugation at 1,100 g for 5 min, resuspend the organisms in an equal volume of Z buffer and place in an ice bath to check the _OD600 value of each sample.4. Two reaction tubes (1 ml each) for each sample:

(1) 100 μl of cell suspension mixed with 900 μl of Z buffer.

(2) 50 μl cell suspension mixed with 950 μl Z buffer.5. In each sample reaction tube, add 1 drop of 0.1% SDS and 2 drops of chloroform with a Pasteur pipette, vortex and oscillate for 10-15 s, and equilibrate for 15 min in a 30°C water bath.6. Add 0.2 ml of 4 mg/ml OPNG, oscillate on a vortex mixer for 5 s, place in a 30°C water bath and start the timer.

7. When the solution turns yellow ( _OD420=0.3~0.7), add 0.5 ml 1 mol/l _Na2CO3 to abort the reaction and record the time of aborting the reaction.8. 1 100 g Centrifuge the cells for 5 min Determine the _OD420 and _OD550 of the supernatant.

9. Calculate the galactosidase activity (in U) according to the following equation

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