Beta-Galactosidase Assay (A better Miller) - Revision history

Andrew T. Martens: /* Background */ It is 1024 amino acids.

Andrew T. Martens — Mon, 27 Aug 2012 23:20:13 GMT

Background: It is 1024 amino acids.

←Older revision		Revision as of 23:20, 27 August 2012
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	{{back to protocols}}		{{back to protocols}}
	==Background==		==Background==
-	[[Beta-galactosidase\|β-Galactosidase]] is encoded by the <i>lacZ</i> gene of the <i>lac</i> operon in <i>E. coli</i>. It is a large (120 kDa, ~~>1000~~ amino acids) protein that forms a tetramer. The enzyme's function in the cell is to cleave lactose to glucose and galactose so that they can be used as carbon/energy sources. The synthetic compound o-nitrophenyl-β-D-galactoside (ONPG) is also recognized as a substrate and cleaved to yield galactose and o-nitrophenol which has a yellow color. When ONPG is in excess over the enzyme in a reaction, the production of o-nitrophenol per unit time is proportional to the concentration of β-Galactosidase; thus, the production of yellow color can be used to determine enzyme concentration.	+	[[Beta-galactosidase\|β-Galactosidase]] is encoded by the <i>lacZ</i> gene of the <i>lac</i> operon in <i>E. coli</i>. It is a large (120 kDa, [http://www.ecocyc.org/ECOLI/NEW-IMAGE?type=ENZYME&object=BETAGALACTOSID-CPLX 1024 amino acids]) protein that forms a tetramer. The enzyme's function in the cell is to cleave lactose to glucose and galactose so that they can be used as carbon/energy sources. The synthetic compound o-nitrophenyl-β-D-galactoside (ONPG) is also recognized as a substrate and cleaved to yield galactose and o-nitrophenol which has a yellow color. When ONPG is in excess over the enzyme in a reaction, the production of o-nitrophenol per unit time is proportional to the concentration of β-Galactosidase; thus, the production of yellow color can be used to determine enzyme concentration.

	So, why do we care? Usually, experiments are designed so that the β-Galactosidase concentration in the cell is a readout for some aspect of a system being studied. For example, an investigator may fuse a promoter to the <i>lacZ</i> gene and use β-Gal levels as a readout for promoter activity under various conditions. In 1972, Jeffrey Miller published "Experiments in Molecular Genetics" which contained a protocol for determining the amount of β-Gal with ONPG. Because of this, ONPG/β-Gal assays are referred to as "Miller" assays, and a standardized amount of β-Gal activity is a "Miller Unit".		So, why do we care? Usually, experiments are designed so that the β-Galactosidase concentration in the cell is a readout for some aspect of a system being studied. For example, an investigator may fuse a promoter to the <i>lacZ</i> gene and use β-Gal levels as a readout for promoter activity under various conditions. In 1972, Jeffrey Miller published "Experiments in Molecular Genetics" which contained a protocol for determining the amount of β-Gal with ONPG. Because of this, ONPG/β-Gal assays are referred to as "Miller" assays, and a standardized amount of β-Gal activity is a "Miller Unit".

Torsten Waldminghaus at 16:59, 23 February 2009

Torsten Waldminghaus — Mon, 23 Feb 2009 16:59:11 GMT

←Older revision		Revision as of 16:59, 23 February 2009
Line 1:		Line 1:
		+	{{back to protocols}}
	==Background==		==Background==
	[[Beta-galactosidase\|β-Galactosidase]] is encoded by the <i>lacZ</i> gene of the <i>lac</i> operon in <i>E. coli</i>. It is a large (120 kDa, >1000 amino acids) protein that forms a tetramer. The enzyme's function in the cell is to cleave lactose to glucose and galactose so that they can be used as carbon/energy sources. The synthetic compound o-nitrophenyl-β-D-galactoside (ONPG) is also recognized as a substrate and cleaved to yield galactose and o-nitrophenol which has a yellow color. When ONPG is in excess over the enzyme in a reaction, the production of o-nitrophenol per unit time is proportional to the concentration of β-Galactosidase; thus, the production of yellow color can be used to determine enzyme concentration.		[[Beta-galactosidase\|β-Galactosidase]] is encoded by the <i>lacZ</i> gene of the <i>lac</i> operon in <i>E. coli</i>. It is a large (120 kDa, >1000 amino acids) protein that forms a tetramer. The enzyme's function in the cell is to cleave lactose to glucose and galactose so that they can be used as carbon/energy sources. The synthetic compound o-nitrophenyl-β-D-galactoside (ONPG) is also recognized as a substrate and cleaved to yield galactose and o-nitrophenol which has a yellow color. When ONPG is in excess over the enzyme in a reaction, the production of o-nitrophenol per unit time is proportional to the concentration of β-Galactosidase; thus, the production of yellow color can be used to determine enzyme concentration.

Reshma P. Shetty at 16:28, 15 October 2007

Reshma P. Shetty — Mon, 15 Oct 2007 16:28:16 GMT

←Older revision		Revision as of 16:28, 15 October 2007
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	==Comments on the assay==		==Comments on the assay==
		+	*'''[[User:Reshma P. Shetty\|Reshma]] 11:28, 15 October 2007 (CDT)''': Miller recommends a culture with OD600 = 0.28 to 0.70. However, he claims that overnight cultures can also be used but that exponentially growing cells give more precise assays <cite>Miller-1992</cite>.

	* When is the reaction done? I never found a good answer for this in the literature. If I let a reaction go to completion, I measured an Abs<sub>420</sub> of ~2-3. Of course, this is out of the reliable range of the spectrophotometer, but it gives an indication of how far the reaction can go. I got reproducible data when the yellow color was just detectable before adding the stop solution up to about the color of LB broth before stopping. Remember, you need the substrate to saturate the enzyme during the course of the reaction, so don't let them go too far. I routinely make three separate measurements for each culture and average them.		* When is the reaction done? I never found a good answer for this in the literature. If I let a reaction go to completion, I measured an Abs<sub>420</sub> of ~2-3. Of course, this is out of the reliable range of the spectrophotometer, but it gives an indication of how far the reaction can go. I got reproducible data when the yellow color was just detectable before adding the stop solution up to about the color of LB broth before stopping. Remember, you need the substrate to saturate the enzyme during the course of the reaction, so don't let them go too far. I routinely make three separate measurements for each culture and average them.
		+	**'''[[User:Reshma P. Shetty\|Reshma]] 11:28, 15 October 2007 (CDT)''': Miller recommends that the OD420nm reading should ideally be 0.6-0.9 <cite>Miller-1992</cite>.

	* Frequently, people use disposable plastic cuvettes to measure both the culture turbidity and the yellow o-nitropenol. It is my experience that the disposable cuvetees have HORRIBLE optics: yes, they are clear, but the light path is pathetically distorted (just look through one at a distant object). This is not a big problem if the same cuvette is used for the blank and the sample and it is oriented in the same way in the spectrophotometer. The problem arises when many different samples are measured in different cuvettes. The values can can vary GREATLY even for the same culture measured in different cuvettes. I recommend using either a high quality glass or quartz cuvette, or measuring the samples in a plate reader using flat-bottomed 96 well plates. I have found my error in pipetting 150 μL of culture for Abs<sub>600</sub> measurements was WAY less than using disposable cuvettes. Of course, the turbidity measured should be calibrated to cells/mL as with a 1cm cuvette.		* Frequently, people use disposable plastic cuvettes to measure both the culture turbidity and the yellow o-nitropenol. It is my experience that the disposable cuvetees have HORRIBLE optics: yes, they are clear, but the light path is pathetically distorted (just look through one at a distant object). This is not a big problem if the same cuvette is used for the blank and the sample and it is oriented in the same way in the spectrophotometer. The problem arises when many different samples are measured in different cuvettes. The values can can vary GREATLY even for the same culture measured in different cuvettes. I recommend using either a high quality glass or quartz cuvette, or measuring the samples in a plate reader using flat-bottomed 96 well plates. I have found my error in pipetting 150 μL of culture for Abs<sub>600</sub> measurements was WAY less than using disposable cuvettes. Of course, the turbidity measured should be calibrated to cells/mL as with a 1cm cuvette.
Line 100:		Line 102:
	#Miller-1972 isbn=0879691069		#Miller-1972 isbn=0879691069
	// (original Miller assay)		// (original Miller assay)
		+	#Miller-1992 isbn=0879693495
	</biblio>		</biblio>

Reshma P. Shetty: /* References */

Reshma P. Shetty — Mon, 15 Oct 2007 14:41:25 GMT

References

←Older revision		Revision as of 14:41, 15 October 2007
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	<biblio>		<biblio>
	#Zhang-JBC-1995 pmid=7538113		#Zhang-JBC-1995 pmid=7538113
		+	// (from which this assay was derived)
		+	#Miller-1972 isbn=0879691069
		+	// (original Miller assay)
	</biblio>		</biblio>

Reshma P. Shetty: /* Recipes */

Reshma P. Shetty — Wed, 10 Oct 2007 17:00:33 GMT

Recipes

←Older revision		Revision as of 17:00, 10 October 2007
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	===Substrate solution===		===Substrate solution===

-	You need 600 μL per ~~reaction~~.	+	You need 600 μL per sample.
-		+	<b>
-	*<b>60 mM Na<sub>2</sub>HPO<sub>4</sub>	+	*60 mM Na<sub>2</sub>HPO<sub>4</sub>
	*40 mM NaH<sub>2</sub>PO<sub>4</sub>		*40 mM NaH<sub>2</sub>PO<sub>4</sub>
	*1 mg/mL o-nitrophenyl-β-D-Galactoside (ONPG)		*1 mg/mL o-nitrophenyl-β-D-Galactoside (ONPG)
-	*2.7 μL/mL β-mercaptoethanol</b>	+	*2.7 μL/mL β-mercaptoethanol
		+	</b>

	(The Zhang protocol also has 20 μg/mL CTAB and 10 μg/mL deoxycholate. I leave these out figuring that there is still plenty from the permeabilization solution and, if they ain't dead yet, they ain't gonna be.)		(The Zhang protocol also has 20 μg/mL CTAB and 10 μg/mL deoxycholate. I leave these out figuring that there is still plenty from the permeabilization solution and, if they ain't dead yet, they ain't gonna be.)

	===Stop solution===		===Stop solution===
-	*~~<b>~~ 1 M Na<sub>2</sub>CO<sub>3</sub> ~~</b>~~	+
		+	You need 700 μL per sample.
		+
		+	*'''1 M Sodium Carbonate (Na<sub>2</sub>CO<sub>3</sub>)'''

	The high pH of the stop solution denatures the β-Gal and approximately doubles the yellow color of the reaction.		The high pH of the stop solution denatures the β-Gal and approximately doubles the yellow color of the reaction.

Reshma P. Shetty: /* References */

Reshma P. Shetty — Mon, 17 Sep 2007 22:29:51 GMT

References

←Older revision		Revision as of 22:29, 17 September 2007
Line 91:		Line 91:

	==References==		==References==
		+	<biblio>
	#Zhang-JBC-1995 pmid=7538113		#Zhang-JBC-1995 pmid=7538113
		+	</biblio>
		+

	[[User:Smoore\|Smoore]] 18:04, 30 Aug 2005 (EDT)		[[User:Smoore\|Smoore]] 18:04, 30 Aug 2005 (EDT)

Reshma P. Shetty: just prettying stuff up to improve readability, please roll back if you don't like it

Reshma P. Shetty — Mon, 17 Sep 2007 22:29:20 GMT

just prettying stuff up to improve readability, please roll back if you don't like it

←Older revision		Revision as of 22:29, 17 September 2007
Line 26:		Line 26:
	In short, the protocol consists of measuring the cell density of a culture of bacteria (Abs<sub>600</sub>), then removing an aliquot of the cells from the cuvette and mixing them with a "permeabilization" solution that contains detergent which disrupts the cell membranes (but leaves the β-Gal intact). This kills the cells and stops translation. After incubation, an ONPG "substrate" solution is added and the yellow color allowed to develop. A "stop" solution is then added and the absorbance of o-nitrophenol is measured.		In short, the protocol consists of measuring the cell density of a culture of bacteria (Abs<sub>600</sub>), then removing an aliquot of the cells from the cuvette and mixing them with a "permeabilization" solution that contains detergent which disrupts the cell membranes (but leaves the β-Gal intact). This kills the cells and stops translation. After incubation, an ONPG "substrate" solution is added and the yellow color allowed to develop. A "stop" solution is then added and the absorbance of o-nitrophenol is measured.

-		+	#Grow cultures under whatever conditions you wish to test.
-	* Grow cultures under whatever conditions you wish to test.	+	#During growth, pre-measure 80 μL aliquots of <b>permeabilization solution </b> into 1.5 mL microfuge tubes and close them.
-		+	#Measure Abs<sub>600</sub> and RECORD IT!
-	* During growth, pre-measure 80 μL aliquots of <b>permeabilization solution </b> into 1.5 mL microfuge tubes and close them.	+	#Remove a 20 μL aliquot of the culture and add it to the 80 μL of permeabilization solution.
-		+
-	* measure Abs<sub>600</sub> and RECORD IT!	+
-		+
-	* Remove a 20 μL aliquot of the culture and add it to the 80 μL of permeabilization solution.	+

	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.

-	* After the last sample is taken, move the samples and the <b>Substrate solution</b> to the 30 °C warm room for 20-30 minutes.	+	#After the last sample is taken, move the samples and the <b>Substrate solution</b> to the 30 °C warm room for 20-30 minutes.
		+	#Add 600 μL of <b>Substrate solution</b> to each tube and NOTE THE TIME OF ADDITION.
		+	#After sufficient color has developed, add 700 μL of <b>Stop solution</b>, mix well, and NOTE THE STOP TIME.
		+	#After stopping the last sample (some may take longer than others, but generally they are done in 30-90 minutes), transfer the tubes to a microfuge and spin for 5-10 minutes at full speed.
		+	#Carefully remove the tubes from the centrifuge and transfer solution from the TOP of the tubes to your cuvette(s). You are trying to avoid having particulate material in the cuvette so that scattering will not influence the reading.
		+	#Record Abs<sub>420</sub>. This should be less than 1 and greater than 0.05. If it's a bit outside of this range, don't sweat it.

-	* Add 600 μL of <b>Substrate solution</b> to each tube and NOTE THE TIME OF ADDITION.	+	Calculate Miller Units as:

-	* ~~After sufficient color has developed, add 700 μL~~ of ~~<b>Stop solution~~</b>~~, mix well, and NOTE THE STOP TIME.~~	+	<math>1000 * \frac{(Abs_{420})}{((Abs_{600} \text{ of culture sampled})(\text{volume } [0.02 \text{ mL}])(\text{reaction time}))}</math>

-	* After stopping the last sample (some may take longer than others, but generally they are done in 30-90 minutes), transfer the tubes to a microfuge and spin for 5-10 minutes at full speed.
-
-	* Carefully remove the tubes from the centrifuge and transfer solution from the TOP of the tubes to your cuvette(s). You are trying to avoid having particulate matertial in the cuvette so that scattering will not influence the reading.
-
-	* Record Abs<sub>420</sub>. This should be less than 1 and greater than 0.05. If it's a bit outside of this range, don't sweat it.
-
-	~~Calculate Miller Units as:~~
-
-	~~1000 * (Abs<sub>420</sub>)/((Abs<sub>600</sub> of culture sampled)(volume [0.02 mL])(reaction time))~~

	==Comments on the assay==		==Comments on the assay==
Line 65:		Line 57:

	*Here's an example of some actual data I obtained using this assay. It was a timecourse experiment. At each time, I removed 1 mL from each of my cultures, measured the OD<sub>600</sub>, took three 20 µL aliquots directly from the cuvette and added each to 80 µL of permeabilization solution. I performed the assay exactly as described above, and all of the samples were kept at room temperature until the timecourse was finished. The OD<sub>600</sub> for these cultures varied from 0.4 to 4 (in my spec) over the course of this experiment and the reaction times for the β-Gal assays varied from 2–25 min. The three individual β-Gal assays for each time point for each culture (red or black symbols) are plotted in the graph to illustrate the reproducibility of the assay within each experiment.--[[User:Kathmc\|Kathleen]]		*Here's an example of some actual data I obtained using this assay. It was a timecourse experiment. At each time, I removed 1 mL from each of my cultures, measured the OD<sub>600</sub>, took three 20 µL aliquots directly from the cuvette and added each to 80 µL of permeabilization solution. I performed the assay exactly as described above, and all of the samples were kept at room temperature until the timecourse was finished. The OD<sub>600</sub> for these cultures varied from 0.4 to 4 (in my spec) over the course of this experiment and the reaction times for the β-Gal assays varied from 2–25 min. The three individual β-Gal assays for each time point for each culture (red or black symbols) are plotted in the graph to illustrate the reproducibility of the assay within each experiment.--[[User:Kathmc\|Kathleen]]
		+
	[[Image:Millergraph.jpg\|500px]]		[[Image:Millergraph.jpg\|500px]]

Line 73:		Line 66:
	You need 80 μL per sample.		You need 80 μL per sample.

-	<b>100 mM dibasic sodium phosphate (Na<sub>2</sub>HPO<sub>4</sub>)</b>	+	*<b>100 mM dibasic sodium phosphate (Na<sub>2</sub>HPO<sub>4</sub>)</b>
-		+	**(The Zhang protocol has 200 mM sodium phosphate. I could never get this into solution with the other components, no matter what I tried so I backed it off to 100 mM. I have even used 50 mM with no detectable change.)
-	(~~the~~ Zhang protocol has 200 mM sodium phosphate. I could never get this into solution with the other components, no matter what I tried so I backed it off to 100 mM. I have even used 50 mM with no detectable change)	+	*<b>20 mM KCl</b>
-		+	*<b>2 mM MgSO<sub>4</sub> </b>
-	<b>20 mM KCl</b>	+	*<b> 0.8 mg/mL CTAB (hexadecyltrimethylammonium bromide)</b>
-		+	*<b> 0.4 mg/mL sodium deoxycholate</b>
-	<b>2 mM MgSO<sub>4</sub> </b>	+	*<b> 5.4 μL/mL beta-mercaptoethanol </b>
-		+
-	<b> 0.8 mg/mL CTAB (hexadecyltrimethylammonium bromide)</b>	+
-		+
-	<b> 0.4 mg/mL sodium deoxycholate</b>	+
-		+
-	<b> 5.4 μL/mL beta-mercaptoethanol </b>	+

	===Substrate solution===		===Substrate solution===
Line 91:		Line 78:
	You need 600 μL per reaction.		You need 600 μL per reaction.

-	<b>60 mM Na<sub>2</sub>HPO<sub>4</sub>	+	*<b>60 mM Na<sub>2</sub>HPO<sub>4</sub>
		+	*40 mM NaH<sub>2</sub>PO<sub>4</sub>
		+	*1 mg/mL o-nitrophenyl-β-D-Galactoside (ONPG)
		+	*2.7 μL/mL β-mercaptoethanol</b>

-	~~40 mM NaH<sub>2</sub>PO<sub>4</sub>~~	+	(The Zhang protocol also has 20 μg/mL CTAB and 10 μg/mL deoxycholate. I leave these out figuring that there is still plenty from the permeabilization solution and, if they ain't dead yet, they ain't gonna be.)
-		+
-	~~1 mg/mL o-nitrophenyl-β-D-Galactoside (ONPG)~~	+
-		+
-	~~2.7 μL/mL β-mercaptoethanol</b>~~	+
-		+
-	(The Zhang protocol also has 20 μg/mL CTAB and 10 μg/mL deoxycholate, I leave these out figuring that there is still plenty from the permeabilization solution and, if they ~~aint~~'t dead yet, they ain't gonna be)	+

	===Stop solution===		===Stop solution===
-		+	*<b> 1 M Na<sub>2</sub>CO<sub>3</sub> </b>
-	<b> 1 M Na<sub>2</sub>CO<sub>3</sub> </b>	+

	The high pH of the stop solution denatures the β-Gal and approximately doubles the yellow color of the reaction.		The high pH of the stop solution denatures the β-Gal and approximately doubles the yellow color of the reaction.

		+	==References==
		+	#Zhang-JBC-1995 pmid=7538113

	[[User:Smoore\|Smoore]] 18:04, 30 Aug 2005 (EDT)		[[User:Smoore\|Smoore]] 18:04, 30 Aug 2005 (EDT)

Reshma P. Shetty: /* Background */ tried to make equation slightly more readable

Reshma P. Shetty — Mon, 17 Sep 2007 22:15:14 GMT

Background: tried to make equation slightly more readable

←Older revision		Revision as of 22:15, 17 September 2007
Line 4:		Line 4:
	So, why do we care? Usually, experiments are designed so that the β-Galactosidase concentration in the cell is a readout for some aspect of a system being studied. For example, an investigator may fuse a promoter to the <i>lacZ</i> gene and use β-Gal levels as a readout for promoter activity under various conditions. In 1972, Jeffrey Miller published "Experiments in Molecular Genetics" which contained a protocol for determining the amount of β-Gal with ONPG. Because of this, ONPG/β-Gal assays are referred to as "Miller" assays, and a standardized amount of β-Gal activity is a "Miller Unit".		So, why do we care? Usually, experiments are designed so that the β-Galactosidase concentration in the cell is a readout for some aspect of a system being studied. For example, an investigator may fuse a promoter to the <i>lacZ</i> gene and use β-Gal levels as a readout for promoter activity under various conditions. In 1972, Jeffrey Miller published "Experiments in Molecular Genetics" which contained a protocol for determining the amount of β-Gal with ONPG. Because of this, ONPG/β-Gal assays are referred to as "Miller" assays, and a standardized amount of β-Gal activity is a "Miller Unit".

-	1 Miller Unit = 1000 * (~~Abs<sub>~~420~~</sub>~~ - (1.75~~Abs<sub>~~550~~</sub>~~))/(~~<i>~~t~~</i>~~ ~~<i>~~v~~</i>~~ * ~~Abs<sub>~~600</~~sub~~>)	+	1 Miller Unit = <math> 1000 * \frac{(Abs_{420} - (1.75Abs_{550}))}{(t v * Abs_{600})}</math>

	where:		where:

-	Abs<sub>420</sub> is the absorbance of the yellow o-nitrophenol,	+	*Abs<sub>420</sub> is the absorbance of the yellow o-nitrophenol,
-		+	*Abs<sub>550</sub> is the scatter from cell debris, which, when multiplied by 1.75 approximates the scatter observed at 420nm,
-	Abs<sub>550</sub> is the scatter from cell debris, which, when multiplied by 1.75 approximates the scatter observed at 420nm,	+	*''t'' = reaction time in minutes,
-		+	*''v'' = volume of culture assayed in milliliters,
-	~~<i>~~t~~</i>~~ = reaction time in minutes,	+	*Abs<sub>600</sub>&dagger; reflects cell density.
-		+
-	~~<i>~~v~~</i>~~ = volume of culture assayed in milliliters,	+
-		+
-	Abs<sub>600</sub>&dagger; reflects cell density.	+
-		+

	&dagger;Note that this value is different for each spectrophotometer used and should be calibrated by plating dilutions of known Abs<sub>600</sub> cultures to determine the colony-forming units per Abs<sub>600</sub>.		&dagger;Note that this value is different for each spectrophotometer used and should be calibrated by plating dilutions of known Abs<sub>600</sub> cultures to determine the colony-forming units per Abs<sub>600</sub>.

Reshma P. Shetty: linked beta-galactosidase page

Reshma P. Shetty — Mon, 17 Sep 2007 22:06:14 GMT

linked beta-galactosidase page

←Older revision		Revision as of 22:06, 17 September 2007
Line 1:		Line 1:
	==Background==		==Background==
-	β-Galactosidase is encoded by the <i>lacZ</i> gene of the <i>lac</i> operon in <i>E. coli</i>. It is a large (120 kDa, >1000 amino acids) protein that forms a tetramer. The enzyme's function in the cell is to cleave lactose to glucose and galactose so that they can be used as carbon/energy sources. The synthetic compound o-nitrophenyl-β-D-galactoside (ONPG) is also recognized as a substrate and cleaved to yield galactose and o-nitrophenol which has a yellow color. When ONPG is in excess over the enzyme in a reaction, the production of o-nitrophenol per unit time is proportional to the concentration of β-Galactosidase; thus, the production of yellow color can be used to determine enzyme concentration.	+	[[Beta-galactosidase\|β-Galactosidase]] is encoded by the <i>lacZ</i> gene of the <i>lac</i> operon in <i>E. coli</i>. It is a large (120 kDa, >1000 amino acids) protein that forms a tetramer. The enzyme's function in the cell is to cleave lactose to glucose and galactose so that they can be used as carbon/energy sources. The synthetic compound o-nitrophenyl-β-D-galactoside (ONPG) is also recognized as a substrate and cleaved to yield galactose and o-nitrophenol which has a yellow color. When ONPG is in excess over the enzyme in a reaction, the production of o-nitrophenol per unit time is proportional to the concentration of β-Galactosidase; thus, the production of yellow color can be used to determine enzyme concentration.

	So, why do we care? Usually, experiments are designed so that the β-Galactosidase concentration in the cell is a readout for some aspect of a system being studied. For example, an investigator may fuse a promoter to the <i>lacZ</i> gene and use β-Gal levels as a readout for promoter activity under various conditions. In 1972, Jeffrey Miller published "Experiments in Molecular Genetics" which contained a protocol for determining the amount of β-Gal with ONPG. Because of this, ONPG/β-Gal assays are referred to as "Miller" assays, and a standardized amount of β-Gal activity is a "Miller Unit".		So, why do we care? Usually, experiments are designed so that the β-Galactosidase concentration in the cell is a readout for some aspect of a system being studied. For example, an investigator may fuse a promoter to the <i>lacZ</i> gene and use β-Gal levels as a readout for promoter activity under various conditions. In 1972, Jeffrey Miller published "Experiments in Molecular Genetics" which contained a protocol for determining the amount of β-Gal with ONPG. Because of this, ONPG/β-Gal assays are referred to as "Miller" assays, and a standardized amount of β-Gal activity is a "Miller Unit".

Reshma P. Shetty at 21:19, 8 May 2007

Reshma P. Shetty — Tue, 08 May 2007 21:19:08 GMT

←Older revision		Revision as of 21:19, 8 May 2007
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	[[Category:Protocol]]		[[Category:Protocol]]
-	~~[[Category:In vivo]]~~
	[[Category:Escherichia coli]]		[[Category:Escherichia coli]]