Knight:Beta-galactosidase assay/96 well format

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.

It is a work in progress and has not been tested!!!

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)
• 10% sodium deoxcholate (light-sensitive, stored at 4°C)
• 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
• 8μL 10% sodium deoxycholate
• 10.8 μL beta-mercaptoethanol

(You need 20 μL per sample.)

Substrate solution

For 10mL

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

(You need 150 μL per sample.)

Protocol

1. Grow cultures in a 96 well deep-well plate under whatever conditions you wish to test.
2. During growth
   1. Make permeabilization solution.
   2. Pre-measure 20 μL aliquots of permeabilization solution into a 96 well microplate and cover to reduce evaporation.
3. Aliquot cultures into a 96 well microplate (175 μL per well).
4. Measure Abs₆₀₀ of cultures using plate reader.
5. Remove a 5 μL aliquot of the culture and add it to the 20 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 subtracting the background absorbance later.
6. Make substrate solution.
7. Warm samples and substrate solution to 30°C
8. Start timer counting up.
9. Every 15 secs, add 150 μL of substrate solution to a row of wells.
10. Note the time of addition.
11. Place the plate in the plate reader to measure the A₄₂₀ and A₅₅₀ over 60-90 mins.

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.

References

1. 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)

2. ISBN:0879691069

   (original Miller assay)

3. ISBN:0879693495
4. 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 |

   96 well format

5. Promega β-galactosidase assays (96 well format and standard curves)

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