View source for Knight:Beta-galactosidase assay/96 well format

This protocol is directly derived from [[User:Smoore|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.  

<font color=red>It is a work in progress and has not been tested!!!</font>

==Materials==
*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.
*4M potassium chloride (KCl)
*1M magnesium sulfate (MgSO<sub>4</sub>)
*1% hexadecyltrimethylammonium bromide (CTAB)
*10% sodium deoxcholate (light-sensitive, stored at 4&deg;C)
*1M NaH<sub>2</sub>PO<sub>4</sub>
*o-nitrophenyl-&beta;-D-Galactoside ONPG (solid)
*1M sodium carbonate (Na<sub>2</sub>CO<sub>3</sub>)

See [[Talk:Knight:Beta-galacosidase assay]] for stock solution recipes.

===Permeabilization Solution===
For 2mL:
*400 &mu;L 500 mM Na<sub>2</sub>HPO<sub>4</sub>
*10&mu;L 4M KCl
*4&mu;L 1M MgSO<sub>4</sub>
*160&mu;L 1% CTAB
*8&mu;L 10% sodium deoxycholate
*10.8 &mu;L beta-mercaptoethanol
*1407.2 &mu;L H<sub>2</sub>O
(You need 20 &mu;L per sample.)

===Substrate solution===
For 10mL
*1.2mL 500mM Na<sub>2</sub>HPO<sub>4</sub>
*400&mu;L 1M NaH<sub>2</sub>PO<sub>4</sub>
*10 mg ONPG
*27 &mu;L &beta;-mercaptoethanol

(You need 150 &mu;L per sample.)

==Protocol==
#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 &mu;L aliquots of permeabilization solution into a 96 well microplate and cover to reduce evaporation.
#Aliquot cultures into a 96 well microplate (175 &mu;L per well).
#Measure Abs<sub>600</sub> of cultures using plate reader.
#Remove a 5 &mu;L aliquot of the culture and add it to the 20 &mu;L 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&deg;C
#Start timer counting up.
#Every 15 secs, add 150 &mu;L of substrate solution to a row of wells.
#Note the time of addition.
#Place the plate in the plate reader to measure the A<sub>420</sub> and A<sub>550</sub> over 60-90 mins.

==Controls==
#Compare measured beta-galactosidase activity in plate reader versus that in microfuge tubes to ensure that the plate is not impacting measured &beta;-galactosidase activity.

==Standard curves==
#Make a standard curve in the plate reader of A<sub>420</sub> vs [[ONP|o-nitrophenol]] concentration using a two-fold serial dilution of ONP.
#Make a standard curve in the plate reader of change in A<sub>420</sub> versus time as a function of &beta;-galactosidase concentration.

==References==
<biblio>
#Zhang-JBC-1995 pmid=7538113
// (from which this assay was derived)
#Miller-1972 isbn=0879691069 
// (original Miller assay)
#Miller-1992 isbn=0879693495
#Griffith-2002 pmid=11779182
// 96 well format
#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)
</biblio>

[[Category:Protocol]]
[[Category:Escherichia coli]]