Download - Bmb 496 spring '10
Figure 1: 14 % SDS-PAGE gel + Coomassie staining
28S 28S 39S 55S
13095725543
34
26
17
10
170Labels:
BUK- 012510- 28S-1 28S-31
NXA-012510-39S-1 39S-31
NXA- 012510-55S-1 55S-31
1
31
Trypsin was used for digestion
Mitochondrial ribosome interacting proteins
Database analysis criteria
• Ribosomal and metabolic proteins, except for S4, 3-ketoacyl-CoA thiolase and acetyl-CoA acetyltransferase, 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.
Mitochondrial Ribosome Associated Proteins
• Huseyin’s samples 39S Control
39S RNase
55S Contol
55S RNase
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.
EF-Tu (49.5 kDa)
0h 1h 2h 3h 4h 0h 16.5h
EF-Tu overexpression
EF-G overexpression
Figure 2 : 12 % SDS-PAGE gel + Coomassie Staining
EF-Tu overexpression : Aliquots (1 ml) were collected at 0, 1 ,2, 3, 4 hours and 40, 25, 15, 10, 5 µl were loaded respectively.
EF-G overexpression: Aliquots (1 ml) were collected at 0, 16.5 hours and 40, 5 µl were loaded respectively.
EF-G (83.5 kDa)
EF-Tu and EF-G overexpression ( 3/25/2010)
• EF-Tu overexpression (4 hours at 37°C) was observed.
• EF-G overexpression (16.5 hours at 22°C) was not visible.
Protein Purification (4/1/2010)15
K- Pe
llet
15K-
Soup
E#1
E#2
E#3
E#4
E#5
Bead
s
15K-
Pelle
t15
K-So
up
E#3
E#5
E#4
E#1
E#2
Bead
s
EF-Tu
EF-G
Figure3&4: 12% SDS-PAGE + Coomassie staining. 20 µl of each sample was loaded. Ni-NTA (PerfectPRO) was used for purification. Ni-NTA resin slurry was incubated with soups at 4°C for 2 hours.
• Both proteins were observed to elute at Eluate #3.
• Some of the protein was left in the pellet.
• Purification was repeated with other pellets of the same samples.
EF-Tu and EF-G Purification (4/16/2010)15
K-S
oup
Was
h #1
E#1
E#2
E#3
E#4
E#5
Beads
15K
-Sou
pW
ash
#1E#
1E#
2E#
3E#
4
Beads
E#5
Figure5&6: Western Blot. 20 µl 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 4°C for 2 hours.
EF- TuEF-G
Figure 7: 12 % SDS-PAGE gel + coommasie staining
EF-Tu overexpression: Aliquots (1 ml) were collected at 0,2,4 hours and 40, 15, 5 µl were loaded respectively.
EF-G overexpression : Aliquots ( 1ml) were collected at 0 and 16.5 hours and 40 and 5 µl were loaded respectively.
0h 16.5h 0h 2h 4h
EF-G EF-Tu
EF-Tu and EF- G overexpression (4.12.2010)
EF-Tu (49.5 kDa)
EF-G (83.5 kDa)
0h 16.5h 0h 2h 4h
EF-G EF-Tu
Figure 8: Western Blot