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=== Protein sample production ===
#[[Target selection|NESG target selection]] <br>
#[[DNA cloning protocols|DNA cloning protocols]]<br>
#[[Protein purification|Protein expression and purification protocols]]&nbsp;<br>
#Sample Optimization
##[[Construct optimization]]
##[[Buffer optimization]]
##[[Cofactor optimization]]
#Initial protein analysis
##[[SDS page gel]]
##[[Protein concentration|Protein concentration measurements]]
##[[Oligomerization Status|Assessment of Oligomerization Status]]
###[[Gel filtration and light scattering|gel-filtration and light scattering]]
###[[NMR determined Rotational correlation time]]
##[[MassSpectrometry|Mass spectrum]]
##NMR screening
###[[1D screening|<sup>1</sup>H 1D screening]]
###[[Nhsqc screen|Initial [<sup>15</sup>N,<sup>1</sup>H] HSQC]]<br>
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=== Resonance Assignment  ===
#[[Resonance Assignment/Principles and concepts|Principles and concepts]]
#[[Resonance Assignment/Practical aspects|Practical aspects]]
##Semi-automated protocols
###[[CARA]]
####[[Spin System Identification with CARA|Spin System Identification in 2D 15N-HSQC and 3D HNNCO]]
####[[Backbone Assignment with CARA|Backbone Resonance Assignment]]
####[[HA and HB Assignment with CARA|Assignment of HA and HB Resonances with (4,3)D GFT HABCAB(CO)NHN]]
####Side Chain Assignment
#####[[Aliphatic Side Chain Assignment with CARA|Aliphatic side-chain assignment]]
#####[[Aromatic Side Chain Assignment with CARA|Aromatic side-chain assignment]]
#####[[Amide Side Chain Assignment with CARA|Amide side-chain assignment]]
###[[Sparky]]
###[[XEASY]]
####[[XEASY Spin system identification|Spin system identification]]
####Backbone resonance assignment'''<br>'''
#####GFT-based spectra
######[[HNCACAB/CABCA(CO)NH]]
#####Conventional spectra
######[[HNCACB/CBCA(CO)NH]]
######HNCA/HN(CO)CA
######HNCO/HN(CA)CO
######NOESY/TOCSY
####[[XEASY Side Chain Assignment|Side chain resonance assignment]]
#####Aliphatic
######GFT NMR spectra
#######[[HA and HB Assignment with GFT in XEASY|(4,3)D GFT HABCAB(CO)NHN]]
#######[[Side chain assignment with aliphatic (4,3)D HCCH-COSY in XEASY|(4,3)D GFT HCCH]]
######Conventional spectra
#######HAHB(CO)NH
#######HCCH-COSY
#######HCCH-TOCSY
#######[[Side chain assignment with CN-NOESY in XEASY|Simultaneous NOESY]]
#######(H)CC-TOCSY-(CO)NH
#######H(CC-TOCSY-CO)NH
#####[[Aromatic side chain assignment with Aro-HCCH-COSY in XEASY|Aromatic]]
######GFT-based spectra
######Conventional spectra
#####Other
######Trp e1 NH and d1 CH
######[[Met methyl assignment with NOESY|Met e CH3 ]]
######[[Amide Side Chain assignment with NOESY|Asn d2 and Gln e2 NH2]]
#####NOESY peak integration
##Automated protocols
###[[AutoAssign|AutoAssign]]
###[[AutoAssign WebServer|AutoAssign server]]
###[[Abacus|ABACUS]]
###[[The PINE Server|PINE server]]
##Validation of resonance assignment
###[[AVS|Assignment validation suite (AVS)]]
###[[LACS|Linear analysis of chemical shift (LACS)]]
##[[PDB and BMRB Deposition#Preparing_files_for_BMRB_depostion|Depositing chemical shifts]]
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Revision as of 22:02, 1 December 2009

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Protein sample production

  1. NESG target selection
  2. DNA cloning protocols
  3. Protein expression and purification protocols 
  4. Sample Optimization
    1. Construct optimization
    2. Buffer optimization
    3. Cofactor optimization
  5. Initial protein analysis
    1. SDS page gel
    2. Protein concentration measurements
    3. Assessment of Oligomerization Status
      1. gel-filtration and light scattering
      2. NMR determined Rotational correlation time
    4. Mass spectrum
    5. NMR screening
      1. 1H 1D screening
      2. Initial [15N,1H] HSQC

Resonance Assignment

  1. Principles and concepts
  2. Practical aspects
    1. Semi-automated protocols
      1. CARA
        1. Spin System Identification in 2D 15N-HSQC and 3D HNNCO
        2. Backbone Resonance Assignment
        3. Assignment of HA and HB Resonances with (4,3)D GFT HABCAB(CO)NHN
        4. Side Chain Assignment
          1. Aliphatic side-chain assignment
          2. Aromatic side-chain assignment
          3. Amide side-chain assignment
      2. Sparky
      3. XEASY
        1. Spin system identification
        2. Backbone resonance assignment
          1. GFT-based spectra
            1. HNCACAB/CABCA(CO)NH
          2. Conventional spectra
            1. HNCACB/CBCA(CO)NH
            2. HNCA/HN(CO)CA
            3. HNCO/HN(CA)CO
            4. NOESY/TOCSY
        3. Side chain resonance assignment
          1. Aliphatic
            1. GFT NMR spectra
              1. (4,3)D GFT HABCAB(CO)NHN
              2. (4,3)D GFT HCCH
            2. Conventional spectra
              1. HAHB(CO)NH
              2. HCCH-COSY
              3. HCCH-TOCSY
              4. Simultaneous NOESY
              5. (H)CC-TOCSY-(CO)NH
              6. H(CC-TOCSY-CO)NH
          2. Aromatic
            1. GFT-based spectra
            2. Conventional spectra
          3. Other
            1. Trp e1 NH and d1 CH
            2. Met e CH3
            3. Asn d2 and Gln e2 NH2
          4. NOESY peak integration
    2. Automated protocols
      1. AutoAssign
      2. AutoAssign server
      3. ABACUS
      4. PINE server
    3. Validation of resonance assignment
      1. Assignment validation suite (AVS)
      2. Linear analysis of chemical shift (LACS)
    4. Depositing chemical shifts


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