Download - Bmb 496 spring '10

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Page 1: 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

Page 2: Bmb 496  spring '10

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.

Page 3: Bmb 496  spring '10

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.

Page 4: Bmb 496  spring '10

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.

Page 5: Bmb 496  spring '10

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.

Page 6: Bmb 496  spring '10

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

Page 7: Bmb 496  spring '10

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


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