Knight:Beta-galactosidase assay/96 well format

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(Permeabilization Solution)
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This protocol is an attempt to modify the protocol to 96 well format for assaying many samples in parallel.   
This protocol is an attempt to modify the protocol to 96 well format for assaying many samples in parallel.   
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<font color=red>It is a work in progress and has not been tested!!!</font>
 
==Materials==
==Materials==
 +
*96 deep well plates for growing cultures
 +
**Square wells aerate the cultures better
 +
*96 well plates for the assay itself
 +
**[[User:Kgriff|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.
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*1M magnesium sulfate (MgSO<sub>4</sub>)
*1M magnesium sulfate (MgSO<sub>4</sub>)
*1% hexadecyltrimethylammonium bromide (CTAB)
*1% hexadecyltrimethylammonium bromide (CTAB)
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*10% sodium deoxcholate (light-sensitive, stored at 4&deg;C)
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*1% sodium deoxcholate (light-sensitive, stored at 4&deg;C)
 +
**10% seems to go funky over time
*1M NaH<sub>2</sub>PO<sub>4</sub>
*1M NaH<sub>2</sub>PO<sub>4</sub>
*o-nitrophenyl-&beta;-D-Galactoside ONPG (solid)
*o-nitrophenyl-&beta;-D-Galactoside ONPG (solid)
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*1M sodium carbonate (Na<sub>2</sub>CO<sub>3</sub>)
 
See [[Talk:Knight:Beta-galacosidase assay]] for stock solution recipes.
See [[Talk:Knight:Beta-galacosidase assay]] for stock solution recipes.
===Permeabilization Solution===
===Permeabilization Solution===
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For 2mL:
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For 8mL:
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*400 &mu;L 500 mM Na<sub>2</sub>HPO<sub>4</sub>
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*1.6 mL 500 mM Na<sub>2</sub>HPO<sub>4</sub>
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*10&mu;L 4M KCl
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*40 &mu;L 4M KCl
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*4&mu;L 1M MgSO<sub>4</sub>
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*16 &mu;L 1M MgSO<sub>4</sub>
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*160&mu;L 1% CTAB
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*480 &mu;L 1% CTAB
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*8&mu;L 10% sodium deoxycholate
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*320 &mu;L 1% sodium deoxycholate
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*10.8 &mu;L beta-mercaptoethanol
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*43.2 &mu;L TCEP (it is a more stable reducing agent than &beta;-mercaptoethanol)
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*1407.2 &mu;L H<sub>2</sub>O
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H<sub>2</sub>O to 8mL
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(You need 20 &mu;L per sample.)
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(You need 80 &mu;L per sample.  This is enough for a 96 well plate.)
===Substrate solution===
===Substrate solution===
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For 10mL
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For 15mL
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*1.2mL 500mM Na<sub>2</sub>HPO<sub>4</sub>
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*1.8mL 500mM Na<sub>2</sub>HPO<sub>4</sub>
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*400&mu;L 1M NaH<sub>2</sub>PO<sub>4</sub>
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*600&mu;L 1M NaH<sub>2</sub>PO<sub>4</sub>
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*10 mg ONPG
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*15 mg ONPG
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*27 &mu;L &beta;-mercaptoethanol
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*40.5 &mu;L TCEP (more stable reducing agent than &beta;-mercaptoethanol)
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(You need 150 &mu;L per sample.  This is enough for a 96 well plate.)
-
(You need 150 &mu;L per sample.)
+
==Protocol==
==Protocol==
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#Grow cultures in a 96 well deep-well plate under whatever conditions you wish to test.
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#*Use incubator in 32-322 because plate shaker in 32-314 doesn't hold the right temperature.
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[[Image:Knight96wellbetagalatosidaseassay.JPG|thumb|right|200px|96 well plate after completion of this assay.]]
 +
 
 +
#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
#During growth
##Make permeabilization solution.
##Make permeabilization solution.
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##Pre-measure 20 &mu;L aliquots of permeabilization solution into a 96 well microplate and cover to reduce evaporation.
+
##Pre-measure 80 &mu;L aliquots of permeabilization solution into a 96 well microplate and cover to reduce evaporation (permeabilization plate).
#Aliquot cultures into a 96 well microplate (175 &mu;L per well).
#Aliquot cultures into a 96 well microplate (175 &mu;L per well).
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#Measure Abs<sub>600</sub> of cultures using plate reader.
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#Measure Abs<sub>600</sub> of cultures using plate reader (absorbance plate).
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#Remove a 5 &mu;L aliquot of the culture and add it to the 20 &mu;L of permeabilization solution.
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#Remove a 20 &mu;L 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.
#*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.
#*Also include a blank (solutions-only) sample for subtracting the background absorbance later.
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#Make substrate solution.
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#Once the time course is nearly complete, make substrate solution.
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#Warm samples and substrate solution to 30&deg;C
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#Add 150 &mu;L substrate solution to each well of measurement plate.
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#Start timer counting up.
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#Add 25 &mu;l of permeabilized samples to measurement plate.
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#Every 15 secs, add 150 &mu;L of substrate solution to a row of wells.
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#Place the plate in the plate reader to measure the A<sub>420</sub> over 60-90 mins.
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#Note the time of addition.
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#*The plate reader does not actually have a 420 excitation filter.  So you must use the CFP 430 excitation filter.
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#Place the plate in the plate reader to measure the A<sub>420</sub> and A<sub>550</sub> over 60-90 mins.
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==Controls==
==Controls==
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#Promega [http://molecool.wustl.edu/krolllab/PDFs/B-gal%20assay-Promega.pdf Promega &beta;-galactosidase assays] (96 well format and standard curves)
#Promega [http://molecool.wustl.edu/krolllab/PDFs/B-gal%20assay-Promega.pdf Promega &beta;-galactosidase assays] (96 well format and standard curves)
#Invitrogen [https://www.invitrogen.com/content/sfs/manuals/bgalassay_man.pdf Invitrogen &beta;-galactosidase assays] (96 well format)
#Invitrogen [https://www.invitrogen.com/content/sfs/manuals/bgalassay_man.pdf Invitrogen &beta;-galactosidase assays] (96 well format)
 +
#Thibodeau-Biotechniques-2004 pmid=15038156
</biblio>
</biblio>
[[Category:Protocol]]
[[Category:Protocol]]
[[Category:Escherichia coli]]
[[Category:Escherichia coli]]

Current revision

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.

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)
  • 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)

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
  • 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.)

Substrate solution

For 15mL

  • 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.)

Protocol

96 well plate after completion of this assay.
96 well plate after completion of this assay.
  1. 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.
  2. During growth
    1. Make permeabilization solution.
    2. Pre-measure 80 μL aliquots of permeabilization solution into a 96 well microplate and cover to reduce evaporation (permeabilization plate).
  3. Aliquot cultures into a 96 well microplate (175 μL per well).
  4. Measure Abs600 of cultures using plate reader (absorbance plate).
  5. 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.
  6. Once the time course is nearly complete, make substrate solution.
  7. Add 150 μL substrate solution to each well of measurement plate.
  8. Add 25 μl of permeabilized samples to measurement plate.
  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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