Beta-galactosidase assay-PDF

Materials

• 500mM dibasic sodium phosphate (Na₂HPO₄)
  • 1M Na₂HPO₄ seems to come out of solution in my hands.
• 4M potassium chloride (KCl)
• 1M magnesium sulfate (MgSO₄)
• 1% hexadecyltrimethylammonium bromide (CTAB)
• 1% sodium deoxcholate (light-sensitive, stored at 4°C)
  • I used to use 10% but the stock solution seemed to go funky over time.
• 1M NaH₂PO₄
• o-nitrophenyl-β-D-Galactoside ONPG (solid)
• 1M sodium carbonate (Na₂CO₃)

Permeabilization Solution

For 2mL:

• 400 μL 500 mM Na₂HPO₄
• 10μL 4M KCl
• 4μL 1M MgSO₄
• 160μL 1% CTAB
• 80μL 1% sodium deoxycholate
• 10.8 μL beta-mercaptoethanol

(You need 80 μL per sample.)

Substrate solution

For 10mL

• 1.2mL 500mM Na₂HPO₄
• 400μL 1M NaH₂PO₄
• 10 mg ONPG
• 27 μL β-mercaptoethanol

(You need 600 μL per sample.)

Protocol

1. Grow cultures under whatever conditions you wish to test.
2. During growth
   1. Make permeabilization solution.
   2. Pre-measure 80 μL aliquots of permeabilization solution into 1.5 mL microfuge tubes and close them.
3. Measure Abs₆₀₀ of cultures and RECORD IT!
4. Remove a 20 μL aliquot of the culture and add it to the 80 μL of permeabilization solution.
   • The sample is now stable for several hours. This allows you to perform time-course experiments.
   • Also include a blank (solutions-only) sample for zero’ing the spec later.
5. Make substrate solution.
6. Warm samples and substrate solution to 30°C
7. Start timer counting up.
8. Every 15 secs, add 600 μL of substrate solution to a sample tube.
9. Note the time of addition.
10. After sufficient color has developed, add 700 μL of 1M Na₂CO₃, mix well.
11. Note the stop time.
12. Once all reactions are complete, transfer the tubes to a microfuge and spin for 10 minutes at full speed.
13. Gently remove tubes from centrifuge.
14. Measure the absorbance at 420nm and 550nm. (Use UV-Vis protocol on Nanodrop).

Calculate Miller Units as:

[math]\displaystyle{ 1000 * \frac{(Abs_{420})}{((Abs_{600} \text{ of culture sampled})*(\text{volume } [0.02 \text{ mL}])*(\text{reaction time}))} }[/math]

or

[math]\displaystyle{ 1000 * \frac{(Abs_{420} – 1.75*Abs_{550})}{((Abs_{600} \text{ of culture sampled})*(\text{volume } [0.02 \text{ mL}])*(\text{reaction time}))} }[/math]

where:

• Abs₄₂₀ is the absorbance of the yellow o-nitrophenol,
• Abs₅₅₀ is the scatter from cell debris, which, when multiplied by 1.75 approximates the scatter observed at 420nm,
• t = reaction time in minutes,
• v = volume of culture assayed in milliliters,
• Abs₆₀₀† reflects cell density.

References

1. ISBN:0879693495 [Miller-1992]
2. Zhang X and Bremer H. Control of the Escherichia coli rrnB P1 promoter strength by ppGpp. J Biol Chem. 1995 May 12;270(19):11181-9. DOI:10.1074/jbc.270.19.11181 | PubMed ID:7538113 | HubMed [Zhang-JBC-1995]
   

   (from which this assay was derived)

3. ISBN:0879691069 [Miller-1972]
   

   (original Miller assay)