Knight:Beta-galactosidase assay

This protocol is directly derived from Sean Moore's Beta-Galactosidase Assay (A better Miller). Please go there for the original protocol. This protocol just contains specifics regarding stock solutions I've made.

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)

See Talk:Knight:Beta-galacosidase assay for stock solution recipes.

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.

Comments on the assay

• Reshma 11:28, 15 October 2007 (CDT): Miller recommends a culture with OD600 = 0.28 to 0.70. However, he claims that overnight cultures can also be used but that exponentially growing cells give more precise assays [1].

• When is the reaction done? I never found a good answer for this in the literature. If I let a reaction go to completion, I measured an Abs₄₂₀ of ~2-3. Of course, this is out of the reliable range of the spectrophotometer, but it gives an indication of how far the reaction can go. I got reproducible data when the yellow color was just detectable before adding the stop solution up to about the color of LB broth before stopping. Remember, you need the substrate to saturate the enzyme during the course of the reaction, so don't let them go too far. I routinely make three separate measurements for each culture and average them.
  • Reshma 11:28, 15 October 2007 (CDT): Miller recommends that the OD420nm reading should ideally be 0.6-0.9 [1].

References

1. ISBN:0879693495
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 |

   (from which this assay was derived)

3. ISBN:0879691069

   (original Miller assay)