Protein concentration - Revision history

TheresaRamelot at 20:25, 21 December 2009

2009-12-21T20:25:40Z

← Older revision		Revision as of 20:25, 21 December 2009
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	== Measuring protein concentration using NanoDrop<br> ==		== Measuring protein concentration using NanoDrop<br> ==

	Both Rutger's University and University of Toronto use the UV absorbance at 280 ~~nmr~~ to determine protein concentrations.		Both Rutger's University and University of Toronto use the UV absorbance at 280 nm to determine protein concentrations.

	Absorbance by proteins is primarily due to the aromatic rings of Phe, Tyr, and Trp residues.		Absorbance by proteins is primarily due to the aromatic rings of Phe, Tyr, and Trp residues.
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	=== ===		=== ===

	=== Procedure (used at Rutger's University) ===		=== Procedure (used at Rutger's University) ===
	<pre>concentration (M) = Absorbance at 280 nm / ( extinction coefficient * path length)		<pre>concentration (M) = Absorbance at 280 nm / ( extinction coefficient * path length)
	</pre>		</pre>
	The extinction coefficent has the units M-1 cm-1.  The path length of most UV spectrophotometers is 1 cm.		The extinction coefficent has the units M-1 cm-1.  The path length of most UV spectrophotometers is 1 cm.

			<br>

			With the NanoDrop instrument, the path length is not 1 cm, but the Absorbance at 280 nm is corrected as if the pathlength is 1cm.  So, the equation to use is just:
	With the NanoDrop instrument, the path length is not 1 cm, but the Absorbance at 280 nm is corrected as if the ~~pathlenght~~ is 1cm.  So, the equation to use is just:		<pre>concentration (M) = Absorbance at 280 nm / extinction coefficient </pre>
	<pre>concentration (M) = Absorbance at 280 nm / extinction coefficient </pre>
	<br>		<br>

TheresaRamelot at 20:22, 21 December 2009

2009-12-21T20:22:25Z

← Older revision		Revision as of 20:22, 21 December 2009
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	== Measuring protein concentration using NanoDrop<br> ==		== Measuring protein concentration using NanoDrop<br> ==

			Both Rutger's University and University of Toronto use the UV absorbance at 280 nmr to determine protein concentrations.

	~~Both Rutger's University~~ and ~~University of Toronto use the UV absorbance at 280 nmr to determine protein concentrations~~.		Absorbance by proteins is primarily due to the aromatic rings of Phe, Tyr, and Trp residues.

	~~Absorbance by proteins is due to~~ the ~~aromatic rings~~ of ~~Phe, Tyr, and Trp residues~~.		Extinction coefficients are estimated using the [http://ca.expasy.org/tools/protparam.html ExPASy ProtParam tool] (a description of the equations used for this calculations can be found [http://ca.expasy.org/tools/protparam-doc.html\| here] )

	~~Extinction coefficients are estimated using the Expasy ProtParam tool (a description of the equations used for this calculations can be found [http://ca.expasy.org/tools/protparam-doc.html\| here] )~~		=== ===

	=== ===		=== Procedure (used at Rutger's University) ===
			<pre>concentration (M) = Absorbance at 280 nm / ( extinction coefficient * path length)
			</pre>
			The extinction coefficent has the units M-1 cm-1.  The path length of most UV spectrophotometers is 1 cm.

	~~=== Procedure (used at Rutger's University)<br> ===~~

	~~concentration = Absorbance at 280 nm divided by absorbance coefficient~~

			With the NanoDrop instrument, the path length is not 1 cm, but the Absorbance at 280 nm is corrected as if the pathlenght is 1cm.  So, the equation to use is just:
			<pre>concentration (M) = Absorbance at 280 nm / extinction coefficient </pre>
	<br>		<br>

	Typical protein concentration measurement protocol with NanoDrop-2000 with connected PC computer.		==== Typical protein concentration measurement protocol with NanoDrop-2000 with connected PC computer. ====

	<br>		<br>

TheresaRamelot at 20:02, 21 December 2009

2009-12-21T20:02:59Z

← Older revision		Revision as of 20:02, 21 December 2009
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	== Measuring protein concentration using NanoDrop<br> ==		== Measuring protein concentration using NanoDrop<br> ==


			Both Rutger's University and University of Toronto use the UV absorbance at 280 nmr to determine protein concentrations.

			Absorbance by proteins is due to the aromatic rings of Phe, Tyr, and Trp residues.

			Extinction coefficients are estimated using the Expasy ProtParam tool (a description of the equations used for this calculations can be found [http://ca.expasy.org/tools/protparam-doc.html\| here] )

			=== ===

	=== Procedure (used at Rutger's University)<br> ===		=== Procedure (used at Rutger's University)<br> ===
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	#Wipe the sensors and pipette on 2 μL of your protein sample. Bring down the lever arm.		#Wipe the sensors and pipette on 2 μL of your protein sample. Bring down the lever arm.
	#Click Measure and record the concentration measured.		#Click Measure and record the concentration measured.
	#In order to determine protein concentration, make note of the intensity at 280 nm.		#In order to determine protein concentration, make note of the intensity at 280 nm.
	#To test multiple samples, just wipe the sensor in between measurements with a Kimwipe. Recalibration or re-blanking is not necessary.		#To test multiple samples, just wipe the sensor in between measurements with a Kimwipe. Recalibration or re-blanking is not necessary.
	#Clean the sample surfaces once more after you are finished.		#Clean the sample surfaces once more after you are finished.

	<br> [[Image:Nanodrop.jpg]]		<br> [[Image:Nanodrop.jpg]]

	=== Procedure (University of Toronto)<br> ===		=== Procedure (University of Toronto)<br> ===

Bs59 at 22:00, 10 December 2009

2009-12-10T22:00:04Z

← Older revision		Revision as of 22:00, 10 December 2009
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	#Wipe the sensors and pipette on 2 μL of your protein sample. Bring down the lever arm.		#Wipe the sensors and pipette on 2 μL of your protein sample. Bring down the lever arm.
	#Click Measure and record the concentration measured.		#Click Measure and record the concentration measured.
	#~~For~~ protein, the ~~peak should be~~ at 280 nm		#In order to determine protein concentration, make note of the intensity at 280 nm.
	#To test multiple samples, just wipe the sensor in between measurements with a Kimwipe. Recalibration or re-blanking is not necessary.		#To test multiple samples, just wipe the sensor in between measurements with a Kimwipe. Recalibration or re-blanking is not necessary.
	#Clean the sample surfaces once more after you are finished.		#Clean the sample surfaces once more after you are finished.

TheresaRamelot at 22:26, 4 December 2009

2009-12-04T22:26:16Z

← Older revision		Revision as of 22:26, 4 December 2009
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	<br>		<br>

	Typical protein ~~peak as measured by the~~ NanoDrop on PC		Typical protein concentration measurement protocol with NanoDrop-2000 with connected PC computer.

	<br>		<br>
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	#Clean the sample surfaces once more after you are finished.		#Clean the sample surfaces once more after you are finished.

	<br>		<br> [[Image:Nanodrop.jpg]]
	[[~~image~~: ~~nanodrop~~.jpg]]

	=== Procedure (University of Toronto)<br> ===		=== Procedure (University of Toronto)<br> ===

TheresaRamelot at 22:24, 4 December 2009

2009-12-04T22:24:40Z

← Older revision		Revision as of 22:24, 4 December 2009
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	#Clean the sample surfaces once more after you are finished.		#Clean the sample surfaces once more after you are finished.

	<br>		<br>
			[[image: nanodrop.jpg]]

	=== Procedure (University of Toronto)<br> ===		=== Procedure (University of Toronto)<br> ===

TheresaRamelot at 23:11, 3 December 2009

2009-12-03T23:11:48Z

← Older revision		Revision as of 23:11, 3 December 2009
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	== Measuring protein concentration using ~~Nanodrop~~<br> ==		== Measuring protein concentration using NanoDrop<br> ==

	=== Procedure (used at Rutger's University)<br> ===		=== Procedure (used at Rutger's University)<br> ===
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	<br>		<br>

	Typical protein peak as measured by the ~~Nanodrop~~ on PC		Typical protein peak as measured by the NanoDrop on PC

	<br>		<br>

TheresaRamelot at 23:10, 3 December 2009

2009-12-03T23:10:44Z

← Older revision		Revision as of 23:10, 3 December 2009
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	== Measuring protein concentration using ~~nanodrop~~<br> ==		== Measuring protein concentration using Nanodrop<br> ==

	=== Procedure (used at Rutger's University)<br> ===		=== Procedure (used at Rutger's University)<br> ===
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	#Clean the sample surfaces once more after you are finished.		#Clean the sample surfaces once more after you are finished.

	<br>		<br>

	=== Procedure (University of Toronto)<br> ===		=== Procedure (University of Toronto)<br> ===

TheresaRamelot at 23:10, 3 December 2009

2009-12-03T23:10:27Z

← Older revision		Revision as of 23:10, 3 December 2009
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	<br>		<br>

	== University of Toronto ~~protein concentration measurements.~~<br> ==		=== Procedure (University of Toronto)<br> ===

	~~since~~ july of 2009, we use thermofisher nanodrop1000 to measure the protein absorbance at 280 nm. ~~the~~ molecular weight and theoretical extinction coefficient is calculated using expasy.		Since july of 2009, we use thermofisher nanodrop1000 to measure the protein absorbance at 280 nm.

			The molecular weight and theoretical extinction coefficient are calculated using expasy.

TheresaRamelot at 23:09, 3 December 2009

2009-12-03T23:09:19Z

← Older revision		Revision as of 23:09, 3 December 2009
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	== Measuring protein concentration using nanodrop<br> ==		== Measuring protein concentration using nanodrop<br> ==

	=== Procedure (used at Rutger's University)<br> ===		=== Procedure (used at Rutger's University)<br> ===



	concentration = Absorbance at 280 nm divided by absorbance coefficient		concentration = Absorbance at 280 nm divided by absorbance coefficient
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	#To test multiple samples, just wipe the sensor in between measurements with a Kimwipe. Recalibration or re-blanking is not necessary.		#To test multiple samples, just wipe the sensor in between measurements with a Kimwipe. Recalibration or re-blanking is not necessary.
	#Clean the sample surfaces once more after you are finished.		#Clean the sample surfaces once more after you are finished.

			<br>

	== University of Toronto protein concentration measurements.<br> ==		== University of Toronto protein concentration measurements.<br> ==

	since july of 2009, we use thermofisher nanodrop1000 to measure the protein absorbance at 280 nm. the molecular weight and theoretical extinction coefficient is calculated using expasy.		since july of 2009, we use thermofisher nanodrop1000 to measure the protein absorbance at 280 nm. the molecular weight and theoretical extinction coefficient is calculated using expasy.