Knight:Beta-galactosidase assay/96 well format
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 () 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)
- 1M sodium carbonate (Na2CO3)
See Talk:Knight:Beta-galacosidase assay for stock solution recipes.
- 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.)
- 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.)
- 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.
- During growth
- Make permeabilization solution.
- Pre-measure 80 μL aliquots of permeabilization solution into a 96 well microplate and cover to reduce evaporation (permeabilization plate).
- Aliquot cultures into a 96 well microplate (175 μL per well).
- Measure Abs600 of cultures using plate reader (absorbance plate).
- 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.
- Once the time course is nearly complete, make substrate solution.
- Add 150 μL substrate solution to each well of measurement plate.
- Add 25 μl of permeabilized samples to measurement plate.
- 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.
- 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.
- Make a standard curve in the plate reader of A420 vs o-nitrophenol concentration using a two-fold serial dilution of ONP.
- Make a standard curve in the plate reader of change in A420 versus time as a function of β-galactosidase concentration.
- 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
- 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.
(from which this assay was derived)
- [by] Jeffrey H. Miller. Experiments in molecular genetics. [Cold Spring Harbor, N.Y.] Cold Spring Harbor Laboratory, 1972. ISBN:0879691069
(original Miller assay)
- Jeffrey H. Miller. A short course in bacterial genetics. Plainview, N.Y.: Cold Spring Harbor Laboratory Press, 1992. ISBN:0879693495
- Promega β-galactosidase assays (96 well format and standard curves)
- Invitrogen β-galactosidase assays (96 well format)
- 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.