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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.



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

Permeabilization Solution

For 8mL:

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

Substrate solution

For 15mL

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


96 well plate after completion of this assay.
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.


Error fetching PMID 7538113:
Error fetching PMID 11779182:
Error fetching PMID 15038156:
  1. Error fetching PMID 11779182: [Griffith-2002]
    96 well format

  2. Error fetching PMID 7538113: [Zhang-JBC-1995]
    (from which this assay was derived)

  3. [by] Jeffrey H. Miller. Experiments in molecular genetics. [Cold Spring Harbor, N.Y.] Cold Spring Harbor Laboratory, 1972. isbn:0879691069. [Miller-1972]
    (original Miller assay)

  4. Jeffrey H. Miller. A short course in bacterial genetics. Plainview, N.Y.: Cold Spring Harbor Laboratory Press, 1992. isbn:0879693495. [Miller-1992]
  5. Promega β-galactosidase assays (96 well format and standard curves)


  6. Invitrogen β-galactosidase assays (96 well format)


  7. Error fetching PMID 15038156: [Thibodeau-Biotechniques-2004]
All Medline abstracts: PubMed HubMed