NESG Wiki - User contributions [en]

Talk:NMR determined Rotational correlation time

2009-12-10T22:01:46Z

Bs59: Created page with ' References are in a different format from the other pages.  --~~~~'

 References are in a different format from the other pages.  --[[User:Bs59|BharathWaj]] 22:01, 10 December 2009 (UTC)

Gel filtration and light scattering

2009-12-10T22:00:59Z

Bs59:

=== 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 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. 

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

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

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

Protein concentration

2009-12-10T22:00:04Z

Bs59:

== Measuring protein concentration using NanoDrop ==

=== Procedure (used at Rutger's University) ===

concentration = Absorbance at 280 nm divided by absorbance coefficient

 

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

 

#Before starting the software module, clean the sample surfaces with DI water to remove any dried sample that might be present.
#Alternatively, you can clean the sample surfaces with a Kimwipe moistened with 70% ethanol.
#Open the Nanodrop program and the appropriate module (e.g., Protein).
#Wipe off the top and bottom sensors of the instrument with a Kimwipe. These are just the polished ends of fiber optic cable, so wiping is sufficient to prevent carryover.
#Pipette 2 μL of DD water onto the sensor. Bring down the lever arm.
#Follow the onscreen prompts to calibrate.
#Wipe the sensors and pipette on 2 μL of the corresponding blank (Buffer solution your protein is in). Bring down the lever arm.
#Follow the onscreen prompts to blank.
#Wipe the sensors and pipette on 2 μL of your protein sample. Bring down the lever arm.
#Click Measure and record the concentration measured.
#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.
#Clean the sample surfaces once more after you are finished.

 [[Image:Nanodrop.jpg]]

=== Procedure (University of Toronto) ===

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.

SDS page gel

2009-12-10T21:58:55Z

Bs59:

== Rutger's University SDS Page Gel Protocol ==

 

Each NESG sample is run on an SDS page gel in order to confirm the monomer size and to look for the presence of SDS-resistant oligomers. These gels are also used to confirm sample purity for each of the labeled samples for NMR.

 

=== Invitrogen NuPage Gel Electrophoresis ===

==== I. Sample Preparation ====

#Add 5 μL 4X Reducing Buffer (RB) to 15μL protein solution. Vortex sample to mix.
#Heat samples at 70 °C for 10 mins.
#Spin samples.

==== II. NuPage Gels ====

#Remove precast gels (4-12 % Bis-Tris gels from Invitrogen) from package. Cut open and dump out liquid in sink. Wash outside with MilliQ water. Pull off white stripe at bottom. 
#Lower buffer core into cell and insert gel cassettes on both sides (make sure the shorter well side faces inward). Insert tension wedge and lock in place. (*If only running one gel, be sure to place buffer dam between the core and the tension wedge). 
#Fill the buffer core with 200 mL 1X RB. Make sure this section does NOT leak before adding liquid to the outside. Carefully remove the comb and pipette out any air bubbles. Then, fill outer part of case with 600 mL of RB. 
#Fill the wells with samples: 
#*Add 10 μL MW markers to well #1. 
#*Fill remaining 11 wells with 10 μL of prepared protein sample solution.
#*Fill each well slowly. Try to avoid bubbles in wells that may cause overflow into other wells 
#*When working with 2 gel plates, make sure to label the outside of the container. 
#Match the + and -electrode ends (same colors)
#*Turn on (be sure Range Select is OFF). Set Range to 200V/400mA, then hit Start.
#*Run for 35 min. When done, then turn off. 
#Gel stain:
#*Take gel cassette out of Invitrogen gel box.
#*Fill microwave safe glass container with MilliQ water enough as to immerse gel.
#*Crack open gel cassette and place gel in microwave safe glass container.
#*Microwave gel for two minutes or until water boils.
#*Place container on shaker until cool. Then decant the water. Rinse one more time with MilliQ water.
#*Add Denville Blue Protein Stain to gel container and immerse gel.   
#*Microwave gel for two minutes or until protein stain solution boils and place on shaker until cool.
#*Decant gel stain into waste container, and rinse gel with MilliQ water.
#*Add MilliQ water to immerse gel and microwave for two minutes and then shake until cool.
#*Carefully place gel into plastic gel box filled with water.

An example of a SDS-PAGE gel run with two protein samples is shown here. Lane#1: Marker, Lane#2: MrR70A-NC5, Lane#3: HR3102A-NC5. [[Image:Nupage gel.gif]]

Buffer optimization

2009-12-10T21:54:59Z

Bs59:

Buffer content plays a critical role in protein sample stability. Buffer optimization may be used to improve sample stability to avoid the following issues: 

#slow precipitation
#mixture of folded and unfolded protein
#aggregation problems
#multiple populations (too many peaks) for other reasons
#and many other reasons

 

At Rutger’s University, NMR samples with promising spectra in the initial pH 6.5, 200 mM NaCl buffer are screened by 1H-15N HSQC spectra after exchanging into twelve commonly used screening buffers (listed below). Exchange is performed using by a desalting column and NMR screening is done with a Bruker MicroCryoprobe.<ref>Rossi, P. et. al. (2009). "A microscale protein NMR sample screening pipeline." ''J. Biomol. NMR'', '''in press.</ref> 

==== '''NMR screening buffers:''' ====

pH 7.5, 50 mM Tris, 500 mM NaCl, 500 mM Imidazole

pH 6.5, 20 mM MES, 100 mM NaCl, 5 mM CaCl2, protease inhibitor 1x

pH 5.5, 20 mM NH4OAc, 100 mM NaCl, 5 mM CaCl2, protease inhibitor 1x

pH 4.5, 20mM NH4OAc, 100 mM NaCl, 5mM CaCl2, protease inhibitor 1x

pH 5.0, 50 mM NH4OAc, 50 mM Arginine, protease inhibitor 1x

pH 5.0, 50 mM NH4OAc, 5% CH3CN, protease inhibitor 1x

pH 6.0, 50 mM MES, 50 mM Arginine, protease inhibitor 1x

pH 6.0, 50 mM MES, 5% CH3CN, protease inhibitor 1x

pH 6.5, 25 mM Na2PO4, 450 mM NaCl, 20 mM ZnSO4, protease inhibitor 1x

pH 6.5, 20 mM MES, 100 mM NaCl, 5% CH3CN, protease inhibitor 1x

pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM Arginine, protease inhibitor 1x

pH 6.5, 20 mM MES, 100 mM NaCl, 1% Zwitter

pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM ZnSO4, protease inhibitor 1x

 

All buffers contain 0.02% NaN3, 10 mM DTT (or 1 mM TCEP), and 5% D2O.

 

TCEP is used instead of DTT, when the protein is eluted from the Ni-column.  Otherwise, Ni+2 and DTT form an insoluble brown precipitate.

 

==== '''Abbreviations:''' ====

DTT: Dithiothreitol

Zwitter: ZWITTERAGENT® 3-12 Detergent cat.963015 (CALBIOCHEM)

MES: 2-(''N''-morpholino)ethanesulfonic acid

Tris: tris(hydroxymethyl)aminomethane;

Protease inhibitor: Protease inhibitor cocktail tablets cat. 11836170001 (ROCHE);

TCEP: ''tris''(2-carboxyethyl)phosphine

==== References ====

<references />

Buffer optimization

2009-12-10T21:53:20Z

Bs59:

Buffer optimization may be used to improve a protein sample with: 

#slow precipitation
#mixture of folded and unfolded protein
#aggregation problems
#multiple populations (too many peaks) for other reasons
#and many other reasons

 

At Rutger’s University, NMR samples with promising spectra in the initial pH 6.5, 200 mM NaCl buffer are screened by 1H-15N HSQC spectra after exchanging into twelve commonly used screening buffers (listed below). Exchange is performed using by a desalting column and NMR screening is done with a Bruker MicroCryoprobe.<ref>Rossi, P. et. al. (2009). "A microscale protein NMR sample screening pipeline." ''J. Biomol. NMR'', '''in press.</ref> 

==== '''NMR screening buffers:''' ====

pH 7.5, 50 mM Tris, 500 mM NaCl, 500 mM Imidazole

pH 6.5, 20 mM MES, 100 mM NaCl, 5 mM CaCl2, protease inhibitor 1x

pH 5.5, 20 mM NH4OAc, 100 mM NaCl, 5 mM CaCl2, protease inhibitor 1x

pH 4.5, 20mM NH4OAc, 100 mM NaCl, 5mM CaCl2, protease inhibitor 1x

pH 5.0, 50 mM NH4OAc, 50 mM Arginine, protease inhibitor 1x

pH 5.0, 50 mM NH4OAc, 5% CH3CN, protease inhibitor 1x

pH 6.0, 50 mM MES, 50 mM Arginine, protease inhibitor 1x

pH 6.0, 50 mM MES, 5% CH3CN, protease inhibitor 1x

pH 6.5, 25 mM Na2PO4, 450 mM NaCl, 20 mM ZnSO4, protease inhibitor 1x

pH 6.5, 20 mM MES, 100 mM NaCl, 5% CH3CN, protease inhibitor 1x

pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM Arginine, protease inhibitor 1x

pH 6.5, 20 mM MES, 100 mM NaCl, 1% Zwitter

pH 6.5, 20 mM MES, 100 mM NaCl, 50 mM ZnSO4, protease inhibitor 1x

 

All buffers contain 0.02% NaN3, 10 mM DTT (or 1 mM TCEP), and 5% D2O.

 

TCEP is used instead of DTT, when the protein is eluted from the Ni-column.  Otherwise, Ni+2 and DTT form an insoluble brown precipitate.

 

==== '''Abbreviations:''' ====

DTT: Dithiothreitol

Zwitter: ZWITTERAGENT® 3-12 Detergent cat.963015 (CALBIOCHEM)

MES: 2-(''N''-morpholino)ethanesulfonic acid

Tris: tris(hydroxymethyl)aminomethane;

Protease inhibitor: Protease inhibitor cocktail tablets cat. 11836170001 (ROCHE);

TCEP: ''tris''(2-carboxyethyl)phosphine

==== References ====

<references />

Protein purification

2009-12-10T21:51:52Z

Bs59:

== Samples for NMR ==

For each protein, we usually make the following samples:

*   100% 15N, 100% 13C uniform labeled sample, for resonance assignment and NOE interpretion.
*   100% 15N, 5% 13C labeled sample, for stereospecific assignment of VAL and LEU isopropyl moieties.

For RDC measurement:

*   A secondary 100% 15N, 5% 13C labeled sample, for RDC measurement.

For each protein that exist as dimer in solution an extra sample may be required in addition to the samples above:

*   1:1 unlabeled and 100% 15N, 100% 13C uniformed labeled mixed sample, for intermolecular NOE interpretation.

== Typical Rutgers University NMR Buffers ==

The protein production facility at Rutgers University uses  typical NMR buffers for the initial protein screening.

 

They are <ref>Snyder, D, et.al. (2005). “Comparisons of NMR spectral quality and success in crystallization demonstrate that NMR and X-ray crystallography are complementary methods for small protein structure determination.” ''JACS'', '''127:''' 16505-16511. [http://www.ncbi.nlm.nih.gov/pubmed/16305237 pmid = 16305237] </ref>:

 

*pH 4.5 NMR buffer:  20 mM NH4OAc, 100 mM NaCl, 10 mM DTT, 5 mM CaCl2, 0.02% NaN3, 5% D2O
*pH 5.5 NMR buffer:  20 mM NH4OAc, 100 mM NaCl, 10 mM DTT, 5 mM CaCl2, 0.02% NaN3, 5% D2O
*pH 6.5 NMR buffer:  20 mM MES, 100 mM NaCl, 5 mM CaCl2, 10 mM DTT, 0.02% NaN3, 5% D2O

 

== Typical Rutgers University Protein Purification Protocol ==

''E.coli'' BL21(DE3)are fermented in MJ9 medium <ref>Jansson M, Li YC, Jendeberg L, Anderson S, Montelione GT, Nilsson B (1996) High-level production of uniformly 15N- and 13C-enriched fusion proteins in ''Escherichia coli''. J Biomol NMR 7: 131-141</ref>.

Cell pellets are stored at -20º C.

(1) add ? to the a frozen cell pellet and thaw.

(2) sonicate in ice bath

(3) centrifigure to remove insoluble part

(4) supernatant is added to an AkTAxpressTM system with a His TrapHP column followed by HiLoad16/60 Superdex 75 gel filtration chromatography.

(5) exchange buffer to screening buffer by concentrating, diluting with new buffer, reconcentrating to 0.3 - 1.0 mM with Amicon ultrafiltration concentrator (Millipore). 

== Typical University of Toronto (Arrowsmith proteomics NMR lab) Buffers ==

standard screening NMR buffers are: a5.0n300zd : 10 mM sodium acetate, pH 5.0, 300 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O 

m6.5n450zd: 10 mM MOPS, pH 6.5, 450 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O

t7.7n500zd : 10 mM tris, pH 7.7, 500 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O 

 

final nmr buffer for 13C/15N labelled sample depends on the protein of interest. all nmr buffers always contain : 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail.

(1) if the protein has no cysteine in the sequence, do not bother to add ZnSO4 and DTT (Zn ion will just be a nuisance and deuterated DTT is expensive).

== Typical University of Toronto Protein Purification Protocol ==

before you start, buffers needed:

i015t8.5n500z : 15 mM imidazole, 10 mM tris, pH 8.5, 500 mM NaCl, 10 uM ZnSO4

i030t8.5n500z : 30 mM imidazole, 10 mM tris, pH 8.5, 500 mM NaCl, 10 uM ZnSO4

i500t8.5n500z : 500 mM imidazole, 10 mM tris, pH 8.5, 500 mM NaCl, 10 uM ZnSO4

1M DTT

1M benzamidine 

(1) add 25 mL of i015t8.5n500z into a frozen cell pellet and thaw. 

(2) sonicate in ice bath

(3) spin down cell pellet

(4) transfer supernatant into new falcon tube and add 3 mL of nickel beads

(5) rock the tube for at least 30 minutes in cold.

(6) spin down the beads and discard the supernatant

(7) wash the beads with i015t8.5n500z twice and with i030t8.5n500z twice 

(8) in the final i030t8.5n500z wash,  pour the beads unto gravity filter column 

(9) elute the protein with i500t8.5n500z

(10) add benzamidine, and add DTT 

(11) exchange buffer by concentrating, diluting with new buffer, reconcentrating in a vivaspin concentrator.

 

if it is 13C/15N sample, add step

(9a) put sample in dialysis bag with protease to cut his-tag and dialyse  against  cleavage buffer

(9b) pass the sample through nickel beads again, then follow step (10) above. 

== References ==

<references />

Protein purification

2009-12-10T21:49:53Z

Bs59:

== Samples for NMR ==

For each protein, we usually make the following samples:

*   100% 15N, 100% 13C uniform labeled sample, for resonance assignment and NOE interpretion.
*   100% 15N, 5% 13C labeled sample, for stereospecific assignment of VAL and LEU isopropyl moieties.

For RDC measurement:

*   A secondary 100% 15N, 5% 13C labeled sample, for RDC measurement.

For each protein that exist as dimer in solution an extra sample may be required in addition to the samples above:

*   1:1 unlabeled and 100% 15N, 100% 13C uniformed labeled mixed sample, for intermolecular NOE interpretation.

== Typical Rutgers University NMR Buffers ==

The protein production facility at Rutgers University uses  typical NMR buffers for the initial protein screening.

 

They are <ref>Snyder, D, et.al. (2005). “Comparisons of NMR spectral quality and success in crystallization demonstrate that NMR and X-ray crystallography are complementary methods for small protein structure determination.” ''JACS'', '''127:''' 16505-16511. [http://www.ncbi.nlm.nih.gov/pubmed/16305237 pmid = 16305237] </ref>:

 

*pH 4.5 NMR buffer:  20 mM NH4OAc, 100 mM NaCl, 10 mM DTT, 5 mM CaCl2, 0.02% NaN3, 5% D2O
*pH 5.5 NMR buffer:  20 mM NH4OAc, 100 mM NaCl, 10 mM DTT, 5 mM CaCl2, 0.02% NaN3, 5% D2O
*pH 6.5 NMR buffer:  20 mM MES, 100 mM NaCl, 5 mM CaCl2, 10 mM DTT, 0.02% NaN3, 5% D2O

 

== Typical Rutgers University Protein Purification Protocol ==

''E.coli'' BL21(DE3)are fermented in MJ9 medium <ref>Jansson M, Li YC, Jendeberg L, Anderson S, Montelione GT, Nilsson B (1996) High-level production of uniformly 15N- and 13C-enriched fusion proteins in ''Escherichia coli''. J Biomol NMR 7: 131-141</ref>.

Cell pellets are stored at -20º C.

(1) add ? to the a frozen cell pellet and thaw.

(2) sonicate in ice bath

(3) centrifigure to remove insoluble part

(4) supernatant is added to an AkTAxpressTM system with a His TrapHP column followed by HiLoad16/60 Superdex 75 gel filtration chromatography.

(5) exchange buffer to screening buffer by concentrating, diluting with new buffer, reconcentrating to 0.3 - 1.0 mM with Amicon ultrafiltration concentrator (Millipore). 

== Typical University of Toronto (Arrowsmith proteomics NMR lab) Buffers ==

standard screening NMR buffers are: a5.0n300zd : 10 mM sodium acetate, pH 5.0, 300 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O 

m6.5n450zd: 10 mM MOPS, pH 6.5, 450 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O

t7.7n500zd : 10 mM tris, pH 7.7, 500 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O 

 

final nmr buffer for 13C/15N labelled sample depends on the protein of interest. all nmr buffers always contain : 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail.

(1) if the protein has no cysteine in the sequence, do not bother to add ZnSO4 and DTT (Zn ion will just be a nuisance and deuterated DTT is expensive).

== Typical University of Toronto Protein Purification Protocol ==

before you start, buffers needed:

i015t8.5n500z : 15 mM imidazole, 10 mM tris, pH 8.5, 500 mM NaCl, 10 uM ZnSO4

i030t8.5n500z : 30 mM imidazole, 10 mM tris, pH 8.5, 500 mM NaCl, 10 uM ZnSO4

i500t8.5n500z : 500 mM imidazole, 10 mM tris, pH 8.5, 500 mM NaCl, 10 uM ZnSO4

1M DTT

1M benzamidine 

(1) add 25 mL of i015t8.5n500z into a frozen cell pellet and thaw. 

(2) sonicate in ice bath

(3) spin down cell pellet

(4) transfer supernatant into new falcon tube and add 3 mL of nickel beads

(5) rock the tube for at least 30 minutes in cold.

(6) spin down the beads and discard the supernatant

(7) wash the beads with i015t8.5n500z twice and with i030t8.5n500z twice 

(8) in the final i030t8.5n500z wash,  pour the beads unto gravity filter column 

(9) elute the protein with i500t8.5n500z

(10) add benzamidine, and add DTT 

(11) exchange buffer by concentrating, diluting with new buffer, reconcentrating in a vivaspin concentrator.

 

if it is 13c/15n sample, add step

(9a) put sample in dialysis bag with protease to cut his-tag and dialyse  against  cleavage buffer

(9b) pass the sample through nickel beads again, then follow step (10) above.
 

== References ==

<references />

Talk:Protein purification

2009-12-10T21:48:26Z

Bs59: Created page with 'Under the section "Typical Rutgers University Protein Purification Protocol" point #(1) information missing and is indicated by a (?) -  --~~~~'

Under the section "Typical Rutgers University Protein Purification Protocol" point #(1) information missing and is indicated by a (?) -  --[[User:Bs59|BharathWaj]] 21:48, 10 December 2009 (UTC)

Protein purification

2009-12-10T21:46:12Z

Bs59:

== Samples for NMR ==

For each protein, we usually make the following samples:

*   100% 15N, 100% 13C uniform labeled sample, for resonance assignment and NOE interpretion.
*   100% 15N, 5% 13C labeled sample, for stereospecific assignment of VAL and LEU isopropyl moieties.

For RDC measurement:

*   A secondary 100% 15N, 5% 13C labeled sample, for RDC measurement.

For each protein that exist as dimer in solution an extra sample may be required in addition to the samples above:

*   1:1 unlabeled and 100% 15N, 100% 13C uniformed labeled mixed sample, for intermolecular NOE interpretation.

== Typical Rutgers University NMR Buffers ==

The protein production facility at Rutgers University uses  typical NMR buffers for the initial protein screening.

 

They are <ref>Snyder, D, et.al. (2005). “Comparisons of NMR spectral quality and success in crystallization demonstrate that NMR and X-ray crystallography are complementary methods for small protein structure determination.” ''JACS'', '''127:''' 16505-16511. [http://www.ncbi.nlm.nih.gov/pubmed/16305237 pmid = 16305237] </ref>:

 

*pH 4.5 NMR buffer:  20 mM NH4OAc, 100 mM NaCl, 10 mM DTT, 5 mM CaCl2, 0.02% NaN3, 5% D2O
*pH 5.5 NMR buffer:  20 mM NH4OAc, 100 mM NaCl, 10 mM DTT, 5 mM CaCl2, 0.02% NaN3, 5% D2O
*pH 6.5 NMR buffer:  20 mM MES, 100 mM NaCl, 5 mM CaCl2, 10 mM DTT, 0.02% NaN3, 5% D2O

 

== Typical Rutgers University Protein Purification Protocol ==

''E.coli'' BL21(DE3)are fermented in MJ9 medium <ref>Jansson M, Li YC, Jendeberg L, Anderson S, Montelione GT, Nilsson B (1996) High-level production of uniformly 15N- and 13C-enriched fusion proteins in ''Escherichia coli''. J Biomol NMR 7: 131-141</ref>.

Cell pellets are stored at -20º C.

(1) add ? to the a frozen cell pellet and thaw.

(2) sonicate in ice bath

(3) centrifigure to remove insoluble part

(4) supernatant is added to an AkTAxpressTM system with a His TrapHP column followed by HiLoad16/60 Superdex 75 gel filtration chromatography.

(5) exchange buffer to screening buffer by concentrating, diluting with new buffer, reconcentrating to 0.3 - 1.0 mM with Amicon ultrafiltration concentrator (Millipore). 

== Typical University of Toronto (Arrowsmith proteomics NMR lab) Buffers ==

standard screening NMR buffers are: a5.0n300zd : 10 mM sodium acetate, pH 5.0, 300 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O 

m6.5n450zd: 10 mM MOPS, pH 6.5, 450 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O

t7.7n500zd : 10 mM tris, pH 7.7, 500 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O 

final nmr buffer for 13c/15n labelled sample depends on the protein of interest. all nmr buffers always contain : 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail.

(1) if the protein has no cysteine in the sequence, do not bother to add ZnSO4 and DTT (Zn ion will just be a nuisance and deuterated DTT is expensive).

== Typical University of Toronto Protein Purification Protocol ==

before you start, buffers needed:

i015t8.5n500z : 15 mM imidazole, 10 mM tris, pH 8.5, 500 mM NaCl, 10 uM ZnSO4

i030t8.5n500z : 30 mM imidazole, 10 mM tris, pH 8.5, 500 mM NaCl, 10 uM ZnSO4

i500t8.5n500z : 500 mM imidazole, 10 mM tris, pH 8.5, 500 mM NaCl, 10 uM ZnSO4

1M DTT

1M benzamidine 

(1) add 25 mL of i015t8.5n500z into a frozen cell pellet and thaw. 

(2) sonicate in ice bath

(3) spin down cell pellet

(4) transfer supernatant into new falcon tube and add 3 mL of nickel beads

(5) rock the tube for at least 30 minutes in cold.

(6) spin down the beads and discard the supernatant

(7) wash the beads with i015t8.5n500z twice and with i030t8.5n500z twice 

(8) in the final i030t8.5n500z wash,  pour the beads unto gravity filter column 

(9) elute the protein with i500t8.5n500z

(10) add benzamidine, and add DTT 

(11) exchange buffer by concentrating, diluting with new buffer, reconcentrating in a vivaspin concentrator.

 

if it is 13c/15n sample, add step

(9a) put sample in dialysis bag with protease to cut his-tag and dialyse  against  cleavage buffer

(9b) pass the sample through nickel beads again, then follow step (10) above.
 

== References ==

<references />

Protein purification

2009-12-10T21:45:34Z

Bs59:

== Samples for NMR ==

For each protein, we usually make the following samples:

*   100% 15N, 100% 13C uniform labeled sample, for resonance assignment and NOE interpretion.
*   100% 15N, 5% 13C labeled sample, for stereospecific assignment of VAL and LEU isopropyl moieties.

For RDC measurement:

*   A secondary 100% 15N, 5% 13C labeled sample, for RDC measurement.

For each protein that exist as dimer in solution an extra sample may be required in addition to the samples above:

*   1:1 unlabeled and 100% 15N, 100% 13C uniformed labeled mixed sample, for intermolecular NOE interpretation.

== Typical Rutgers University NMR Buffers ==

The protein production facility at Rutgers University uses  typical NMR buffers for the initial protein screening.

 

They are <ref>Snyder, D, et.al. (2005). “Comparisons of NMR spectral quality and success in crystallization demonstrate that NMR and X-ray crystallography are complementary methods for small protein structure determination.” ''JACS'', '''127:''' 16505-16511. [http://www.ncbi.nlm.nih.gov/pubmed/16305237 pmid = 16305237] </ref>:

 

*pH 4.5 NMR buffer:  20 mM NH4OAc, 100 mM NaCl, 10 mM DTT, 5 mM CaCl2, 0.02% NaN3, 5% D2O
*pH 5.5 NMR buffer:  20 mM NH4OAc, 100 mM NaCl, 10 mM DTT, 5 mM CaCl2, 0.02% NaN3, 5% D2O
*pH 6.5 NMR buffer:  20 mM MES, 100 mM NaCl, 5 mM CaCl2, 10 mM DTT, 0.02% NaN3, 5% D2O

 

== Typical Rutgers University Protein Purification Protocol ==

''E.coli'' BL21(DE3)are fermented in MJ9 medium <ref>Jansson M, Li YC, Jendeberg L, Anderson S, Montelione GT, Nilsson B (1996) High-level production of uniformly 15N- and 13C-enriched fusion proteins in ''Escherichia coli''. J Biomol NMR 7: 131-141</ref>.

Cell pellets are stored at -20º C.

(1) add ? to the a frozen cell pellet and thaw.

(2) sonicate in ice bath

(3) centrifigure to remove insoluble part

(4) supernatant is added to an AkTAxpressTM system with a His TrapHP column followed by HiLoad16/60 Superdex 75 gel filtration chromatography.

(5) exchange buffer to screening buffer by concentrating, diluting with new buffer, reconcentrating to 0.3 - 1.0 mM with Amicon ultrafiltration concentrator (Millipore). 

== Typical University of Toronto (Arrowsmith proteomics NMR lab) Buffers ==

standard screening NMR buffers are: a5.0n300zd : 10 mM sodium acetate, pH 5.0, 300 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O 

m6.5n450zd: 10 mM MOPS, pH 6.5, 450 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O

t7.7n500zd : 10 mM tris, pH 7.7, 500 mM NaCl, 10 uM ZnSO4, 10 mM DTT, 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail, 5% D2O 

final nmr buffer for 13c/15n labelled sample depends on the protein of interest. all nmr buffers always contain : 0.01 % NaN3, 1 mM benzamidine, 1x inhibitor cocktail.

(1) if the protein has no cysteine in the sequence, do not bother to add ZnSO4 and DTT (Zn ion will just be a nuisance and deuterated DTT is expensive).

== Typical University of Toronto Protein Purification Protocol ==

before you start, buffers needed:

i015t8.5n500z : 15 mM imidazole, 10 mM tris, pH 8.5, 500 mM NaCl, 10 uM ZnSO4

i030t8.5n500z : 30 mM imidazole, 10 mM tris, pH 8.5, 500 mM NaCl, 10 uM ZnSO4

i500t8.5n500z : 500 mM imidazole, 10 mM tris, pH 8.5, 500 mM NaCl, 10 uM ZnSO4

1M DTT

1M benzamidine 

(1) add 25 mL of i015t8.5n500z into a frozen cell pellet and thaw. 

(2) sonicate in ice bath

(3) spin down cell pellet

(4) transfer supernatant into new falcon tube and add 3 mL of nickel beads

(5) rock the tube for at least 30 minutes in cold.

(6) spin down the beads and discard the supernatant

(7) wash the beads with i015t8.5n500z twice and with i030t8.5n500z twice 

(8) in the final i030t8.5n500z wash,  pour the beads unto gravity filter column 

(9) elute the protein with i500t8.5n500z

(10) add benzamidine, and add DTT 

(11) exchange buffer by concentrating, diluting with new buffer, reconcentrating in a vivaspin concentrator.

 

if it is 13c/15n sample, add step

(9a) put sample in dialysis bag with protease to cut his-tag and dialyse  against  cleavage buffer

(9b) pass the sample through nickel beads again, then follow step (10) above.
 

== References ==

<references />

DNA cloning protocols

2009-12-10T21:44:09Z

Bs59:

== Typical Rutger's University Cloning Protocol for Target Proteins ==

'''Target selection'''

Protein targets are selected based on target selection criteria. See [[Target selection]] page. 

 

'''Construct design '''

Multiple constructs (typically 2-3) are designed for each full length target and targeted domains within targets.  Constructs are designed with developing automated construct design software which employs homology to the PDB, secondary structure predictions, and disorder predictions.  Predicted signal peptides are removed from auto-generated constructs.  If a target is predicted to be extra-cellular, only constructs with one or no cysteines are generated since extra-cellular proteins often require disulfide bonds for stability and proper folding that are not readily formed with the NESG's ''E. coli'' cytosolic expression systems.  Predicted transmembrane helices are removed from auto-generated constructs.

 

'''PCR primers and vectors '''

PCR primers are designed using the NESG's PCR primer design software ( [http://www-nmr.cabm.rutgers.edu/bioinformatics/Primer_Primer/ Primer Prim’er] <ref>Everett JK, Acton, TB, Montelione, GT.(2004) Primer prim'er: a web baased server for automated primer design. J Struct Funct Genom 5: 13-21. </ref> ) for insertion into pET15 or pET21 (Invitrogen) expression vector derivatives that have amino and carboxy terminal hexa-His tags, respectively. PCR primers are designed to add 15 base pairs to the end of each PCR amplified fragment desinged to pair with vectors poly-cloning region. Fusion of the PCR product and the linearized vector is performed using the Clontech In-FusionTM PCR Cloning System. 

== References ==

<references />

Talk:Target selection

2009-12-10T21:43:23Z

Bs59: Created page with '"70% of its resources to the determination of targeted families selected...": determination implies structure determination? --~~~~ "70% of its resources to the …'

"70% of its resources to the determination of targeted families selected...": determination implies structure determination? --[[User:Bs59|BharathWaj]] 21:43, 10 December 2009 (UTC)

"70% of its resources to the determination..." + "30% to targets nominated by ..." + "30% to its own biomedicaly oriented ...": Total % exceeds 100. --[[User:Bs59|BharathWaj]] 21:43, 10 December 2009 (UTC)