Thursday, December 26, 2024

Beta-galactosidase assay-PDF

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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)

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