ß-galactosidase assays

26 April 2005

Ted Young lab, UW Biochemistry, from Miller 1972

Grow and harvest cells (keep everything sterile)

1. grow 5 mls culture for each assay (large white cap glass tubes, shaking, usually at at 30^o)

• if not maintaining a plasmid, grow in YP + 5% glucose

• if maintaining a plasmid, grow in appropriate SM dropout medium + 5% glucose

• start a 5mls culture and grow at least 6 hours, then dilute, usually in 10-20 mls so they will be OD₆₀₀ = 0.5-1.0 at harvest or derepression

• to calculate dilution, use N₀ = N_t/2^g

N_t = OD₆₀₀ you want at harvest

N₀ = OD₆₀₀ at which to start the culture

g = number of generations culture will go through overnight

2. for “derepressed” DR samples:

• spin 2.5K, 5 min (RC-3B)

• discard supernatant (“sup”) by pouring carefully into the sink and not losing the cell pellet

• wash with 5 mls sterile water (add water, carefully resuspend by vortexing, spin again and discard sup)

• resuspend pellet in same type and amount of medium but 0.05% glucose

• grow at 30^o, shaking

3. when cells are ready (e.g. after 6 hours of DR), harvest by spinning 2.5K, 5 min

• remove sup as above, keep pellet in the tube on ice

• proceed to step 5 or freeze

4. to freeze:

• resuspend in 1 ml cold Z-buffer with 18% glycerol, transfer to 1.5 ml Eppendorf tube, spin in a microfuge and remove sup

• freeze pellet on dry ice, transfer to –70^o

• thaw on ice when ready to use

ß-gal assay (not necessary to keep sterile)

before starting:

• turn on water bath to 28^o

• add 2.7µl ß-mercaptoethanol per ml Z-buffer to 2-3 mls per sample;

• thaw ONPG

• have everything ready by the water bath: timer, ONPG, pipettors and tips, NaCO3, beta-gal experiment sheet to write down times

5. resuspend cell pellet in 120µl cold Z-buffer, vortex, keep on ice

6. follow directions starting with step 2 on the Beta-gal assay experiment sheet

• for vol. #1, use 5µl, be sure to vortex before taking out the cells because they settle

• for vol. #2, use 1-5µl if you expect high activity (e.g. derepressed wildtype, constitutive mutant, etc.), 25-50µl if you expect medium activity, 100µl if you expect low activity (e.g. repressed or activation-defective mutant)

• for step 3 set them up in short glass white-capped tubes (you can remove the caps if it's easier)

• for step 6, add NaCO3 when a faint yellow color developes

7. use an Excel spreadsheet to calculate ß-galactosidase activity in Miller units as directed on the worksheet

8. If A420 readings are higher than 1, go back and re-do the samples using fewer cells.

solutions

• Z buffer (on the shelf, remember to add ß-ME)

Na₂HPO₄•7H₂O 16.1g

NaH₂PO₄•7H₂O 5.5 g

MgSO₄•7H₂O 0.246 g

dH₂O ~800 ml

pH to 7.0 using HCl

bring to 1L with dH₂O

autoclave

• 1M Na₂CO₃ (sodium carbonate): 53g/500 mls H2O

• ONPG: 200 mg/50 mls H₂O, can be frozen at -20

• 0.1% SDS

• chloroform

Nataly's method:

6. read OD₆₀₀ of a 10X dilution in water (make a 1ml sample for the J429 spectrophotometer)

7. in small glass tubes, make each sample 2 OD₆₀₀ in 1 ml with Z-buffer

8. add 2 drops of 0.1% SDS and 3 drops of CHCl₃ to each sample with a Pasteur pipet

9. vortex 10 sec

10. incubate 15 minutes at 28^o

11. add 0.2 ml ONPG, vortex, return to water bath

• start a timer when adding ONPG to the first tube and add to the rest at 1 minute intervals

12. when faint yellow color develops (lighter than a yellow tip), add 0.5 ml Na₂CO₃ and note the time

13. when all samples are finished, pellet 3K, 5 minutes

14. measure A₄₂₀