Knight:Beta-galactosidase assay/96 well format

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(Permeabilization Solution)
(Permeabilization Solution)
Line 30: Line 30:
*480 μL 1% CTAB
*480 μL 1% CTAB
*32 μL 10% sodium deoxycholate
*32 μL 10% sodium deoxycholate
-
*43.2 μL β-mercaptoethanol
+
*43.2 μL TCEP (it is a more stable reducing agent than β-mercaptoethanol)
-
**maybe should try substituting TCEP for β-mercaptoethanol since it is a more stable reducing agent
+
H<sub>2</sub>O to 8mL
H<sub>2</sub>O to 8mL
(You need 80 &mu;L per sample.  This is enough for a 96 well plate.)
(You need 80 &mu;L per sample.  This is enough for a 96 well plate.)

Revision as of 14:56, 27 November 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 8mL:

  • 1.6 mL 500 mM Na2HPO4
  • 40 μL 4M KCl
  • 16 μL 1M MgSO4
  • 480 μL 1% CTAB
  • 32 μL 10% 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.)

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. Add 150 μL substrate solution to each well.
  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.

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]
  7. Thibodeau SA, Fang R, and Joung JK. . pmid:15038156. PubMed HubMed [Thibodeau-Biotechniques-2004]
All Medline abstracts: PubMed HubMed
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