Knight:Beta-galactosidase assay/96 well format

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This protocol is directly derived from Sean Moore's Beta-Galactosidase Assay (A better Miller). Please go there for the original protocol.

This protocol is an attempt to modify the protocol to 96 well format for assaying many samples in parallel.


  • 96 deep well plates for growing cultures
    • Square wells aerate the cultures better
  • 96 well plates for the assay itself
    • Kevin Griffith ([1]) uses Marsh brand (MP-9091) 96 well plates available through ThermoFisher Scientific (but not available on the website). This plate is a polystyrene plate that has a max well volume of 0.3ml. The wells are a flat bottom design. These plates are not coated or treated. The MP-9091 is packaged as 100 plates per case.
  • 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)
    • 10% seems to go funky over time
  • 1M NaH2PO4
  • o-nitrophenyl-β-D-Galactoside ONPG (solid)

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

Permeabilization Solution

For 8mL:

  • 1.6 mL 500 mM Na2HPO4
  • 40 μL 4M KCl
  • 16 μL 1M MgSO4
  • 480 μL 1% CTAB
  • 320 μL 1% sodium deoxycholate
  • 43.2 μL TCEP (it is a more stable reducing agent than β-mercaptoethanol)

H2O to 8mL (You need 80 μL per sample. This is enough for a 96 well plate.)

Substrate solution

For 15mL

  • 1.8mL 500mM Na2HPO4
  • 600μL 1M NaH2PO4
  • 15 mg ONPG
  • 40.5 μL TCEP (more stable reducing agent than β-mercaptoethanol)

(You need 150 μL per sample. This is enough for a 96 well plate.)


96 well plate after completion of this assay.
  1. Grow cultures in tubes under whatever conditions you wish to test.
    • 96 well plates did not give me as good of growth as tubes.
    • If growing in 96 well plates, use incubator in 32-322 because plate shaker in 32-314 doesn't hold the right temperature.
  2. During growth
    1. Make permeabilization solution.
    2. Pre-measure 80 μL aliquots of permeabilization solution into a 96 well microplate and cover to reduce evaporation (permeabilization plate).
  3. Aliquot cultures into a 96 well microplate (175 μL per well).
  4. Measure Abs600 of cultures using plate reader (absorbance plate).
  5. Remove a 20 μL aliquot of each well of the absorbance plate and add it to the corresponding well of the permeabilization plate.
    • The sample is now stable for several hours. This allows you to perform time-course experiments.
    • Also include a blank (solutions-only) sample for subtracting the background absorbance later.
  6. Once the time course is nearly complete, make substrate solution.
  7. Add 150 μL substrate solution to each well of measurement plate.
  8. Add 25 μl of permeabilized samples to measurement plate.
  9. Place the plate in the plate reader to measure the A420 over 60-90 mins.
    • The plate reader does not actually have a 420 excitation filter. So you must use the CFP 430 excitation filter.


  1. Compare measured beta-galactosidase activity in plate reader versus that in microfuge tubes to ensure that the plate is not impacting measured β-galactosidase activity.

Standard curves

  1. Make a standard curve in the plate reader of A420 vs o-nitrophenol concentration using a two-fold serial dilution of ONP.
  2. Make a standard curve in the plate reader of change in A420 versus time as a function of β-galactosidase concentration.


  1. Griffith KL and Wolf RE Jr. Measuring beta-galactosidase activity in bacteria: cell growth, permeabilization, and enzyme assays in 96-well arrays. Biochem Biophys Res Commun. 2002 Jan 11;290(1):397-402. DOI:10.1006/bbrc.2001.6152 | PubMed ID:11779182 | HubMed [Griffith-2002]

    96 well format

  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)

  4. ISBN:0879693495 [Miller-1992]
  5. Promega β-galactosidase assays (96 well format and standard curves)

  6. [Invitrogen]
  7. Thibodeau SA, Fang R, and Joung JK. High-throughput beta-galactosidase assay for bacterial cell-based reporter systems. Biotechniques. 2004 Mar;36(3):410-5. DOI:10.2144/04363BM07 | PubMed ID:15038156 | HubMed [Thibodeau-Biotechniques-2004]

All Medline abstracts: PubMed | HubMed