Gel filtration and light scattering - Revision history

Alex at 17:07, 16 December 2009

2009-12-16T17:07:10Z

← Older revision		Revision as of 17:07, 16 December 2009
Line 1:		Line 1:
	~~=== Gel filtration and static light scattering at Rutger's University ===~~

	==== Gel filtration ====		==== Gel filtration ====

	Gel filtration is performed at 4 °C on a Superdex 75 (26/60) ~~column~~ equilibrated with the corresponding NMR buffer at a flow rate of 2.5 ml/min. The column is calibrated with LMW gel filtration calibration kit (17-0442-01) from GE Healthcare. A calibration curve is prepared by measuring the elution volumes of several standards, calculating their corresponding K<sub>av</sub> values and plotting their K<sub>av</sub> values versus the logarithm of their molecular weight. The molecular weight of a protein is determined from the calibration curve once its K<sub>av</sub> value is calculated from its measured elution volume.		Gel filtration is performed at 4 °C on a Superdex 75 (26/60) (CABM Rutgers) or Superdex 75 Hiload 16/60 (U of Toronto) equilibrated with the corresponding NMR buffer at a flow rate of 2.5 ml/min. The column is calibrated with LMW gel filtration calibration kit (17-0442-01) from GE Healthcare. A calibration curve is prepared by measuring the elution volumes of several standards, calculating their corresponding K<sub>av</sub> values and plotting their K<sub>av</sub> values versus the logarithm of their molecular weight. The molecular weight of a protein is determined from the calibration curve once its K<sub>av</sub> value is calculated from its measured elution volume.

	<math>K_{av}=\frac{V_e-V_0}{V_t-V_0}</math>		<math>K_{av}=\frac{V_e-V_0}{V_t-V_0}</math>
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	The MW of protein can also be determined by analytical size exclusion chromatography combined with multi-angle light scattering (SEC-MALS). The measurements are performed on an Agilent 1100 HPLC system (Agilent) connected to a tri-angle light scattering detector and a differential refractometer (miniDAWN Tristar and Optilab, Wyatt Technology). A Shodex KW-802.5 column (Shodex) is equilibrated with the corresponding NMR buffer at a flow rate of 0.5 ml/min. A volume of 30 μl is injected. Data is processed using ASTRA software (Wyatt Technology) assuming a specific refractive index increment (dn/dc) of 0.185 ml/g. To determine the detector delay volumes and the normalization coefficients for the MALS detector, a BSA sample (Sigma) is used as a reference.		The MW of protein can also be determined by analytical size exclusion chromatography combined with multi-angle light scattering (SEC-MALS). The measurements are performed on an Agilent 1100 HPLC system (Agilent) connected to a tri-angle light scattering detector and a differential refractometer (miniDAWN Tristar and Optilab, Wyatt Technology). A Shodex KW-802.5 column (Shodex) is equilibrated with the corresponding NMR buffer at a flow rate of 0.5 ml/min. A volume of 30 μl is injected. Data is processed using ASTRA software (Wyatt Technology) assuming a specific refractive index increment (dn/dc) of 0.185 ml/g. To determine the detector delay volumes and the normalization coefficients for the MALS detector, a BSA sample (Sigma) is used as a reference.

	~~=== Gel filtration at University of Toronto ===~~

	~~Gel filtration chromatography is used to determine the protein oligomerization state in solution. <br>~~

	~~equipment: biologic duo flow; column: Superdex 75 Hiload 16/60~~

Alex: /* Gel filtration and static light scattering at Rutger's University */

2009-12-16T17:02:51Z

Gel filtration and static light scattering at Rutger's University

← Older revision		Revision as of 17:02, 16 December 2009
Line 1:		Line 1:
	=== Gel filtration and static light scattering at Rutger's University ===		=== Gel filtration and static light scattering at Rutger's University ===

	~~<br> '''Oligomerization Analysis by~~ Gel ~~Filtration''' <br> The molecular weight of protein is determined by gel~~ filtration. It is performed at 4 °C on a Superdex 75 (26/60) column equilibrated with the NMR buffer pH 6.5 at a flow rate of 2.5 ml/min. The column is calibrated with LMW gel filtration calibration kit (17-0442-01) from GE Healthcare. A calibration curve is prepared by measuring the elution volumes of several standards, calculating their corresponding Kav values and plotting their Kav values versus the logarithm of their molecular weight. The molecular weight of a protein is determined from the calibration curve once its Kav value is calculated from its measured elution volume. <br>		==== Gel filtration ====

	~~Kav =~~(~~Ve - Vo~~)/(Vt - Vo)		Gel filtration is performed at 4 °C on a Superdex 75 (26/60) column equilibrated with the corresponding NMR buffer at a flow rate of 2.5 ml/min. The column is calibrated with LMW gel filtration calibration kit (17-0442-01) from GE Healthcare. A calibration curve is prepared by measuring the elution volumes of several standards, calculating their corresponding K<sub>av</sub> values and plotting their K<sub>av</sub> values versus the logarithm of their molecular weight. The molecular weight of a protein is determined from the calibration curve once its K<sub>av</sub> value is calculated from its measured elution volume.
	~~where Ve =~~ elution ~~volume for~~ the protein
	~~Vo = column void volume =~~ elution volume ~~for Blue Dextran 2000~~
	~~Vt = total bed volume~~

	<br> ~~The oligomerization state of protein is estimated from the value of MW(Measured)~~/~~MW(Predicted). Typically a protein momomer or dimer will run "larger" than predicted, but never "smaller".~~		<math>K_{av}=\frac{V_e-V_0}{V_t-V_0}</math>

	~~'''Static Light Scattering Analysis'''~~ <br> The MW of protein is also determined by analytical size exclusion chromatography combined with multi-angle light scattering (SEC-MALS). The measurements ~~were~~ performed on an Agilent 1100 HPLC system (Agilent) connected to a tri-angle light scattering detector and a differential refractometer (miniDAWN Tristar and Optilab, Wyatt Technology). A Shodex KW-802.5 column (Shodex) ~~was~~ equilibrated ~~in 10 mM MES, pH 6.5, 100 mM NaCl, 5.0 mM CaCl2, 10 mM DTT, and 0.02% NaN3~~ at a flow rate of 0.5 ml/min. A volume of 30 μl ~~was~~ injected. Data ~~were~~ processed using ASTRA software (Wyatt Technology) assuming a specific refractive index increment (dn/dc) of 0.185 ml/g. To determine the detector delay volumes and the normalization coefficients for the MALS detector, a BSA sample (Sigma) ~~was~~ used as a reference. ~~<br>~~		where V<sub>e</sub> is the elution volume for the protein, V<sub>0</sub> is the column void volume (elution volume for Blue Dextran 2000) and V<sub>t</sub> is the total bed volume.

			The oligomerization state of protein is estimated from the ratio of measured to calculated molecular weight. Typically, a protein momomer or dimer will run "larger" than predicted, but never "smaller".

			==== Static light scattering ====

			The MW of protein can also be determined by analytical size exclusion chromatography combined with multi-angle light scattering (SEC-MALS). The measurements are performed on an Agilent 1100 HPLC system (Agilent) connected to a tri-angle light scattering detector and a differential refractometer (miniDAWN Tristar and Optilab, Wyatt Technology). A Shodex KW-802.5 column (Shodex) is equilibrated with the corresponding NMR buffer at a flow rate of 0.5 ml/min. A volume of 30 μl is injected. Data is processed using ASTRA software (Wyatt Technology) assuming a specific refractive index increment (dn/dc) of 0.185 ml/g. To determine the detector delay volumes and the normalization coefficients for the MALS detector, a BSA sample (Sigma) is used as a reference.

	=== Gel filtration at University of Toronto ===		=== Gel filtration at University of Toronto ===

Bs59 at 22:00, 10 December 2009

2009-12-10T22:00:59Z

← Older revision		Revision as of 22:00, 10 December 2009
Line 4:		Line 4:

	Kav =(Ve - Vo)/(Vt - Vo)		Kav =(Ve - Vo)/(Vt - Vo)
	where Ve = elution volume for the protein		where Ve = elution volume for the protein
	Vo = column void volume = elution volume for Blue Dextran 2000		Vo = column void volume = elution volume for Blue Dextran 2000
	Vt = total bed volume		Vt = total bed volume

	<br> The oligomerization state of protein is estimated from the value of MW(Measured)/MW(Predicted). Typically a protein momomer or dimer will run "larger" than predicted, but never "smaller".		<br> The oligomerization state of protein is estimated from the value of MW(Measured)/MW(Predicted). Typically a protein momomer or dimer will run "larger" than predicted, but never "smaller".

	'''Static Light Scattering Analysis''' <br> The MW of protein ~~was~~ also determined by analytical size exclusion chromatography combined with multi-angle light scattering (SEC-MALS). The measurements were performed on an Agilent 1100 HPLC system (Agilent) connected to a tri-angle light scattering detector and a differential refractometer (miniDAWN Tristar and Optilab, Wyatt Technology). A Shodex KW-802.5 column (Shodex) was equilibrated in 10 mM MES, pH 6.5, 100 mM NaCl, 5.0 mM CaCl2, 10 mM DTT, and 0.02% NaN3 at a flow rate of 0.5 ml/min. A volume of 30 μl was injected. Data were processed using ASTRA software (Wyatt Technology) assuming a specific refractive index increment (dn/dc) of 0.185 ml/g. To determine the detector delay volumes and the normalization coefficients for the MALS detector, a BSA sample (Sigma) was used as a reference. <br>		'''Static Light Scattering Analysis''' <br> The MW of protein is also determined by analytical size exclusion chromatography combined with multi-angle light scattering (SEC-MALS). The measurements were performed on an Agilent 1100 HPLC system (Agilent) connected to a tri-angle light scattering detector and a differential refractometer (miniDAWN Tristar and Optilab, Wyatt Technology). A Shodex KW-802.5 column (Shodex) was equilibrated in 10 mM MES, pH 6.5, 100 mM NaCl, 5.0 mM CaCl2, 10 mM DTT, and 0.02% NaN3 at a flow rate of 0.5 ml/min. A volume of 30 μl was injected. Data were processed using ASTRA software (Wyatt Technology) assuming a specific refractive index increment (dn/dc) of 0.185 ml/g. To determine the detector delay volumes and the normalization coefficients for the MALS detector, a BSA sample (Sigma) was used as a reference. <br>



	=== Gel filtration at University of Toronto ===		=== Gel filtration at University of Toronto ===

TheresaRamelot at 18:43, 29 November 2009

2009-11-29T18:43:40Z

← Older revision		Revision as of 18:43, 29 November 2009
Line 8:		Line 8:
	Vt = total bed volume		Vt = total bed volume

	<br>		<br> The oligomerization state of protein is estimated from the value of MW(Measured)/MW(Predicted). Typically a protein momomer or dimer will run "larger" than predicted, but never "smaller".
	The oligomerization state of protein is estimated from the value of MW(Measured)/MW(Predicted). Typically a protein momomer or dimer will run "larger" than predicted, but never "smaller".

	'''Static Light Scattering Analysis'''		'''Static Light Scattering Analysis''' <br> The MW of protein was also determined by analytical size exclusion chromatography combined with multi-angle light scattering (SEC-MALS). The measurements were performed on an Agilent 1100 HPLC system (Agilent) connected to a tri-angle light scattering detector and a differential refractometer (miniDAWN Tristar and Optilab, Wyatt Technology). A Shodex KW-802.5 column (Shodex) was equilibrated in 10 mM MES, pH 6.5, 100 mM NaCl, 5.0 mM CaCl2, 10 mM DTT, and 0.02% NaN3 at a flow rate of 0.5 ml/min. A volume of 30 μl was injected. Data were processed using ASTRA software (Wyatt Technology) assuming a specific refractive index increment (dn/dc) of 0.185 ml/g. To determine the detector delay volumes and the normalization coefficients for the MALS detector, a BSA sample (Sigma) was used as a reference. <br>
	<br>
	The MW of protein was also determined by analytical size exclusion chromatography combined with multi-angle light scattering (SEC-MALS). The measurements were performed on an Agilent 1100 HPLC system (Agilent) connected to a tri-angle light scattering detector and a differential refractometer (miniDAWN Tristar and Optilab, Wyatt Technology). A Shodex KW-802.5 column (Shodex) was equilibrated in 10 mM MES, pH 6.5, 100 mM NaCl, 5.0 mM CaCl2, 10 mM DTT, and 0.02% NaN3 at a flow rate of 0.5 ml/min. A volume of 30 μl was injected. Data were processed using ASTRA software (Wyatt Technology) assuming a specific refractive index increment (dn/dc) of 0.185 ml/g. To determine the detector delay volumes and the normalization coefficients for the MALS detector, a BSA sample (Sigma) was used as a reference.
	<br>
			=== Gel filtration at University of Toronto ===

			Gel filtration chromatography is used to determine the protein oligomerization state in solution. <br>

			equipment: biologic duo flow; column: Superdex 75 Hiload 16/60

TheresaRamelot: Created page with '=== Gel filtration and static light scattering at Rutger's University ===
'''Oligomerization Analysis by Gel Filtration'''
The molecular weight of protein is determin…'

2009-11-12T14:31:34Z

Created page with '=== Gel filtration and static light scattering at Rutger's University === '''Oligomerization Analysis by Gel Filtration''' The molecular weight of protein is determin…'

New page

=== Gel filtration and static light scattering at Rutger's University ===

 '''Oligomerization Analysis by Gel Filtration''' The molecular weight of protein is determined by gel filtration. It is performed at 4 °C on a Superdex 75 (26/60) column equilibrated with the NMR buffer pH 6.5 at a flow rate of 2.5 ml/min. The column is calibrated with LMW gel filtration calibration kit (17-0442-01) from GE Healthcare. A calibration curve is prepared by measuring the elution volumes of several standards, calculating their corresponding Kav values and plotting their Kav values versus the logarithm of their molecular weight. The molecular weight of a protein is determined from the calibration curve once its Kav value is calculated from its measured elution volume. 

Kav =(Ve - Vo)/(Vt - Vo)
where Ve = elution volume for the protein
Vo = column void volume = elution volume for Blue Dextran 2000
Vt = total bed volume

 
The oligomerization state of protein is estimated from the value of MW(Measured)/MW(Predicted). Typically a protein momomer or dimer will run "larger" than predicted, but never "smaller".

'''Static Light Scattering Analysis'''
 
The MW of protein was also determined by analytical size exclusion chromatography combined with multi-angle light scattering (SEC-MALS). The measurements were performed on an Agilent 1100 HPLC system (Agilent) connected to a tri-angle light scattering detector and a differential refractometer (miniDAWN Tristar and Optilab, Wyatt Technology). A Shodex KW-802.5 column (Shodex) was equilibrated in 10 mM MES, pH 6.5, 100 mM NaCl, 5.0 mM CaCl2, 10 mM DTT, and 0.02% NaN3 at a flow rate of 0.5 ml/min. A volume of 30 μl was injected. Data were processed using ASTRA software (Wyatt Technology) assuming a specific refractive index increment (dn/dc) of 0.185 ml/g. To determine the detector delay volumes and the normalization coefficients for the MALS detector, a BSA sample (Sigma) was used as a reference.

Gel filtration and light scattering - Revision history

Alex at 17:07, 16 December 2009

Alex: /* Gel filtration and static light scattering at Rutger's University */

Bs59 at 22:00, 10 December 2009

TheresaRamelot at 18:43, 29 November 2009

TheresaRamelot: Created page with '=== Gel filtration and static light scattering at Rutger's University === '''Oligomerization Analysis by Gel Filtration''' The molecular weight of protein is determin…'

TheresaRamelot: Created page with '=== Gel filtration and static light scattering at Rutger's University ===
'''Oligomerization Analysis by Gel Filtration'''
The molecular weight of protein is determin…'