bmb 496 spring '10
out of 7
Post on 06-Aug-2015
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- 1. Mitochondrial ribosome interacting proteins
BUK- 012510- 28S-1 28S-31
NXA- 012510-55S-1 55S-31
Trypsin was used for digestion
Figure 1: 14 % SDS-PAGE gel + Coomassie staining
- 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. Mitochondrial Ribosome Associated Proteins
Huseyins samples 39SControl
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. EF-Tu and EF-G overexpression ( 3/25/2010)
0h 1h2h3h4h0h 16.5h
EF-G (83.5 kDa)
- EF-Tuoverexpression (4 hours at 37C) was observed.
- 5. EF-G overexpression (16.5 hours at 22C) was not visible.
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.
- 6. Protein Purification (4/1/2010)
- 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.
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.
- 9. EF-Tu and EF-G Purification (4/16/2010)
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.
EF-Tu and EF- G overexpression (4.12.2010)
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
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