Knight:Beta-galactosidase assay/96 well format

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(Permeabilization Solution)
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==Materials==
==Materials==
 +
*96 deep well plates for growing cultures
 +
**Square wells aerate the cultures better
 +
*96 well plates for the assay itself
 +
**Kevin Griffith (<cite>Griffith-2002</cite>) 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 (Na<sub>2</sub>HPO<sub>4</sub>)
*500mM dibasic sodium phosphate (Na<sub>2</sub>HPO<sub>4</sub>)
**1M Na<sub>2</sub>HPO<sub>4</sub> seems to come out of solution in my hands.
**1M Na<sub>2</sub>HPO<sub>4</sub> seems to come out of solution in my hands.

Revision as of 10:08, 26 October 2007

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!!!

Contents

Materials

  • 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)
  • 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 β-mercaptoethanol
    • maybe should try substituting TCEP for β-mercaptoethanol since it is a more stable reducing agent
  • 1407.2 μL H2O

(You need 20 μL per sample.)

Substrate solution

For 10mL

  • 1.2mL 500mM Na2HPO4
  • 400μL 1M NaH2PO4
  • 10 mg ONPG
  • 27 μL β-mercaptoethanol
    • maybe should try substituting TCEP for β-mercaptoethanol since it is a more stable reducing agent

(You need 150 μL per sample.)

Protocol

  1. 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.
  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.

Controls

  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.

References

  1. Griffith KL and Wolf RE Jr. . pmid:11779182. PubMed HubMed [Griffith-2002]
    96 well format

  2. Zhang X and Bremer H. . pmid:7538113. PubMed HubMed [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) [Promega]
  6. Invitrogen β-galactosidase assays (96 well format) [Invitrogen]
All Medline abstracts: PubMed HubMed
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