1D NMR Titrations - Revision history

Wiki Administrator at 20:25, 21 July 2022

2022-07-21T20:25:01Z

← Older revision		Revision as of 20:25, 21 July 2022
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	* It has been found that the best protein concentration range is 0.00 uM to 40.0 uM to get a complete binding curve for the majority of ligands. This is only for HSA and is most likely different for different proteins.		* It has been found that the best protein concentration range is 0.00 uM to 40.0 uM to get a complete binding curve for the majority of ligands. This is only for HSA and is most likely different for different proteins.
	* Solution Human Serum Albumin is only good for about fourteen days, make sure to write down the date made and make new sample every 14days as needed		* Solution Human Serum Albumin is only good for about fourteen days, make sure to write down the date made and make new sample every 14days as needed

	~~[[category:Protocols\|NMR Titrations]]~~
	[[category:NMR_Usage\|NMR Titrations]]		[[category:NMR_Usage\|NMR Titrations]]
	[[category:Sample_Preparation\|NMR Titrations]]		[[category:Sample_Preparation\|NMR Titrations]]

Mjeppesen: LearningSorting

2021-09-11T04:18:15Z

LearningSorting

← Older revision		Revision as of 04:18, 11 September 2021
Line 164:		Line 164:
	* Solution Human Serum Albumin is only good for about fourteen days, make sure to write down the date made and make new sample every 14days as needed		* Solution Human Serum Albumin is only good for about fourteen days, make sure to write down the date made and make new sample every 14days as needed

	[[category:Protocols\|~~Protocols~~]]		[[category:Protocols\|NMR Titrations]]
	[[category:NMR_Usage]]		[[category:NMR_Usage\|NMR Titrations]]
	[[category:Sample_Preparation]]		[[category:Sample_Preparation\|NMR Titrations]]

Mjeppesen: Added to Category

2021-09-11T03:36:26Z

Added to Category

← Older revision		Revision as of 03:36, 11 September 2021
Line 163:		Line 163:
	* It has been found that the best protein concentration range is 0.00 uM to 40.0 uM to get a complete binding curve for the majority of ligands. This is only for HSA and is most likely different for different proteins.		* It has been found that the best protein concentration range is 0.00 uM to 40.0 uM to get a complete binding curve for the majority of ligands. This is only for HSA and is most likely different for different proteins.
	* Solution Human Serum Albumin is only good for about fourteen days, make sure to write down the date made and make new sample every 14days as needed		* Solution Human Serum Albumin is only good for about fourteen days, make sure to write down the date made and make new sample every 14days as needed

			[[category:Protocols\|Protocols]]
			[[category:NMR_Usage]]
			[[category:Sample_Preparation]]

Jcatazaro at 15:19, 20 March 2017

2017-03-20T15:19:49Z

← Older revision		Revision as of 15:19, 20 March 2017
Line 163:		Line 163:
	* It has been found that the best protein concentration range is 0.00 uM to 40.0 uM to get a complete binding curve for the majority of ligands. This is only for HSA and is most likely different for different proteins.		* It has been found that the best protein concentration range is 0.00 uM to 40.0 uM to get a complete binding curve for the majority of ligands. This is only for HSA and is most likely different for different proteins.
	* Solution Human Serum Albumin is only good for about fourteen days, make sure to write down the date made and make new sample every 14days as needed		* Solution Human Serum Albumin is only good for about fourteen days, make sure to write down the date made and make new sample every 14days as needed

	~~[[Category:Protocols]]~~

Wiki Administrator: 1 revision: Protocols

2012-03-13T00:02:22Z

1 revision: Protocols

New page

== Protein Stock Preparation ==
# Make 2.0 mM (2000 uM) HSA stock solution
## Dissolve 130.8 mg of HSA (MW = 65400 g) in 1.0 mL D2O
# Make a set of serial dilutions starting with the 2.0 mM stock (see table 1) (NOTE: at these stock solutions it only takes 10.0 uL of protein sample to give the desired protein concentration in the NMR tube)
# Spin Samples down in mini centrifuge
# Press short button until max speed then release
# Measure the absorbance and generate a standard curve for the new samples.
# Make 10X dilutions (10.0 uL HSA and 990 uL D2O)

'''Table 1:'''
{| class="wikitable"
|-
! Sample No.
! NMR [HSA] (uM)
! [HSA] (uM)
! Volume (n+1) sample (uL)
! Volume of D2O (uL)
|-
| 1
| 0.000
| 0.00
| 0
| 0
|-
| 2
| 0.100
| 5.00
| 500
| 500
|-
| 3
| 0.200
| 10.00
| 500
| 500
|-
| 4
| 0.400
| 20.00
| 667
| 333
|-
| 5
| 0.600
| 30.00
| 750
| 250
|-
| 6
| 0.800
| 40.00
| 800
| 200
|-
| 7
| 1.000
| 50.00
| 500
| 500
|-
| 8
| 2.000
| 100.00
| 667
| 333
|-
| 9
| 3.000
| 150.00
| 750
| 250
|-
| 10
| 4.000
| 200.00
| 667
| 333
|-
| 11
| 6.000
| 300.00
| 750
| 250
|-
| 12
| 8.000
| 400.00
| 800
| 200
|-
| 13
| 10.00
| 500.00
| 833
| 167
|-
| 14
| 12.00
| 600.00
| 857
| 143
|-
| 15
| 14.00
| 700.00
| 875
| 125
|-
| 16
| 16.00
| 800.00
| 889
| 111
|-
| 17
| 18.00
| 900.00
| 900
| 100
|-
| 18
| 20.00
| 1000.00
| 667
| 333
|-
| 19
| 30.00
| 1500.00
| 750
| 250
|-
| 20
| 40.00
| 2000.00
| 130.8 mg
| 1 mL
|}

== Ligand Stock Preparation ==
# Make 50.0 mM Potassium Phosphate buffer at pH 7.0 in D2O
## For 500.0 mL total solution, measure 2.177 g K2HPO4 (MW = 174.18 g/mol) and 1.70 g KH2PO4 (MW = 136.09g/mol) in separate 250.0 mL volumetric flasks and fill to line with 99.9% D2O.
## Spike with 500 uL of TMSP from 10.0 uM stock solution
# Make 500.0 uL fresh ligand stocks at 20.0 mM concentration
## Calculate the mass of ligand needed and dissolve in 500.0 uL of 100% DMSO
# Make 11.0 mL titration stocks
## Single ligands: In Falcon tube add, in order, 10.5 mL Potassium Phosphate buffer, 440 uL 100% DMSO and finally 55 uL of fresh ligand stock.
## Ligand Mixtures: In a Falcon tube add in order: 10.390 mL Potassium Phosphate buffer, 440 uL 100% DMSO and finally 55μL of each fresh ligand stock solution (The 2 non-binders Choline Bromide and Uridine-5’-monophosphate plus the binding ligand)
# Vortex the titration stock solutions to mix

== NMR Sample Preparation ==
# Label 20 1.5 uL centrifuge tubes with the ligand and protein concentration
# To each tube add 490 uL of the ligand titration stock solution
# To each tube add 10 uL of the specific protein stock to the intended sample (i.e. add 10 uL of 400 uM stock to get protein concentration in NMR tube of 8 uM)
# Spin samples down in mini-centrifuge to make sure all sample is in the bottom of tube
# Pipette sample out of centrifuge tubes and into NMR tubes making sure not to get any bubbles
# Run samples on NMR using the ''zgesgp'' pulse program (''1D-FASTNMR'' parameter set) or other comparable method for water pre-saturation

=== Important Notes ===
* This is a general method for preparing samples for NMR titrations and can be amended as needed.
* It has been found that the best protein concentration range is 0.00 uM to 40.0 uM to get a complete binding curve for the majority of ligands. This is only for HSA and is most likely different for different proteins.
* Solution Human Serum Albumin is only good for about fourteen days, make sure to write down the date made and make new sample every 14days as needed

[[Category:Protocols]]