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.
It is a work in progress and has not been tested!!!
- 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.
- 400 μL 500 mM Na2HPO4
- 10μL 4M KCl
- 4μL 1M MgSO4
- 160μL 1% CTAB
- 8μL 10% sodium deoxycholate
- 10.8 μL beta-mercaptoethanol
- 1407.2 μL H2O
(You need 20 μL per sample.)
- 1.2mL 500mM Na2HPO4
- 400μL 1M NaH2PO4
- 10 mg ONPG
- 27 μL β-mercaptoethanol
(You need 150 μL per sample.)
- Grow cultures in a 96 well deep-well plate under whatever conditions you wish to test.
- Use incubator in 32-322 because plate shaker in 32-314 doesn't hold the right temperature.
- During growth
- Make permeabilization solution.
- Pre-measure 20 μL aliquots of permeabilization solution into a 96 well microplate and cover to reduce evaporation.
- Aliquot cultures into a 96 well microplate (175 μL per well).
- Measure Abs600 of cultures using plate reader.
- 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.
- Make substrate solution.
- Warm samples and substrate solution to 30°C
- Start timer counting up.
- Every 15 secs, add 150 μL of substrate solution to a row of wells.
- Note the time of addition.
- Place the plate in the plate reader to measure the A420 and A550 over 60-90 mins.
- 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.
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