bmb 496 spring '10

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  1. 1. Mitochondrial ribosome interacting proteins
    28S 28S39S55S
    1
    170
    130
    Labels:
    BUK- 012510- 28S-1 28S-31
    NXA-012510-39S-1 39S-31
    NXA- 012510-55S-1 55S-31
    95
    72
    55
    43
    34
    26
    Trypsin was used for digestion
    17
    10
    31
    Figure 1: 14 % SDS-PAGE gel + Coomassie staining
  2. 2. Database analysis criteria
    Ribosomal and metabolic proteins, except for S4, 3-ketoacyl-CoA thiolase and acetyl-CoAacetyltransferase, were not taken into consideration.
    Proteins found at least in one of the subunits (28S, 39S) and in the intact ribosome were noted.
    For the proteins that were observed in multiple bands, the ones with the highest scores were selected.
    The results that scored lower than 55 and included more than 1 peptide were excluded.
    The results that scored lower than 50 were excluded from the list.
  3. 3. Mitochondrial Ribosome Associated Proteins
    Huseyins samples 39SControl
    39SRNase
    55SContol
    55SRNase
    Database analysis criteria:
    Proteins that were present in control samples and lost in RNase samples were reported as ribosome associated proteins in the list of 39S and 55S.
    MRPs that are present in control samples and increase in RNase samples were reported in the list of 39S and 55S.
    Apoptosis inducing factor (AIF) was investigated by Western Blot by Huseyin.No difference was observed between Control and RNase samples.
  4. 4. EF-Tu and EF-G overexpression ( 3/25/2010)
    EF-Tuoverexpression
    EF-G overexpression
    0h 1h2h3h4h0h 16.5h
    EF-G (83.5 kDa)
    EF-Tu(49.5 kDa)
    • EF-Tuoverexpression (4 hours at 37C) was observed.
    • 5. EF-G overexpression (16.5 hours at 22C) was not visible.
    Figure 2 : 12 % SDS-PAGE gel + Coomassie Staining
    EF-Tuoverexpression : Aliquots (1 ml) were collected at 0, 1 ,2, 3, 4 hours and 40, 25, 15, 10, 5 l were loaded respectively.
    EF-G overexpression: Aliquots (1 ml) were collected at 0, 16.5 hours and 40, 5 lwere loaded respectively.
  5. 6. Protein Purification (4/1/2010)
    Beads
    15K-Soup
    15K- Pellet
    E#3
    E#5
    E#2
    E#1
    E#4
    EF-Tu
    • Both proteins were observed to elute at Eluate #3.
    • 7. Some of the protein was left in the pellet.
    • 8. Purification was repeated with other pellets of the same samples.
    Beads
    15K- Pellet
    15K-Soup
    E#3
    E#5
    E#2
    E#1
    E#4
    EF-G
    Figure3&4: 12% SDS-PAGE + Coomassie staining. 20 l of each sample was loaded. Ni-NTA (PerfectPRO) was used for purification. Ni-NTA resin slurry was incubated with soups at 4C for 2 hours.
  6. 9. EF-Tu and EF-G Purification (4/16/2010)
    Wash #1
    15K-Soup
    Beads
    E#5
    Wash #1
    E#1
    E#4
    E#2
    15K-Soup
    E#5
    E#3
    E#1
    E#4
    Beads
    E#2
    E#3
    EF-G
    EF- Tu
    Figure5&6: Western Blot. 20 l of each sample was loaded to 12% SDS-PAGE gels. Ni-NTA was used for purification (O/N washing). Ni-NTA resin slurry was incubated with soups at 4C for 2 hours.
  7. 10.
    EF-Tu and EF- G overexpression (4.12.2010)
    EF-GEF-Tu
    EF-GEF-Tu
    0h16.5h0h2h 4h
    0h 16.5h 0h 2h4h
    EF-G (83.5 kDa)
    EF-Tu (49.5 kDa)
    Figure 7: 12 % SDS-PAGE gel + coommasie staining
    EF-Tuoverexpression: Aliquots (1 ml) were collected at 0,2,4 hours and 40, 15, 5 l were loaded respectively.
    EF-G overexpression : Aliquots ( 1ml) were collected at 0 and 16.5 hours and 40 and 5 l were loaded respectively.
    Figure 8: Western Blot