6. class single molecule fluor part 2 Flashcards
(73 cards)
1
Q
Single molecule enzymatics
A
2
Q
Single molecule enzymatics dynamics
A
3
Q
Time dependent kcat
A
4
Q
The lipase B from the yeast candida antarctica
A
5
Q
Non fluorescent substrate conversion
A
6
Q
Long observation time possible
A
7
Q
Several conformations are involved
A
8
Q
How many conformations are involved?
A
9
Q
The ‘lazy’ enzyme
A
10
Q
Size of the fluorogenic substrate
A
11
Q
Size of the substrate
A
12
Q
Single molecule enzymatics
A
13
Q
Single molecule enzymatics
A
14
Q
Phospholipases
A
15
Q
Phospholipase A1 studied
A
16
Q
Single molecule enzymatics
A
17
Q
Single molecule enzymatics
first step
A
18
Q
Single molecule enzymatics?
A
19
Q
Water soluble perylenediimide dye (PDI)
A
20
Q
Dual color excitation
A
21
Q
Active PLA1 – High concentration
A
22
Q
Active PLA1 – Low concentration
A
23
Q
Active PLA1 vs inactive
A
24
Q
Single enzyme trajectories
A
25
Image analysis
26
hetero vs Homogeneous track
27
Cummulative distribution functions
28
Enzyme mobility
29
Model of mode of operation
30
Getting closer to cell membranes....
31
Getting closer to nature....
32
Hydrolysis of giant unilamellar vesicles (GUV’s)
33
cytoplasma & er
34
YFP-PLCβ1 – Ca2+ response
35
eGFP-PLCβ1 – Ca2+ response + SPT
36
DNA sequencing & the human genome project
37
DNA sequencing
38
DNA-sequencing history
39
1 st gen: Sanger
40
1 st gen: Sanger method
41
1 st gen: Automated Sanger Method
42
1 st gen: Automated Sanger Method
43
1 st gen: Automated Sanger Method
44
2 nd gen: 454 sequencing
45
2 nd gen: 454 sequencing (Roche®)
46
2 nd gen: 454 sequencing (Roche®)
47
2 nd gen: 454 sequencing (Roche®)
48
2 nd gen: Illumina® (Solexa) sequencing
49
2 nd gen: Illumina® (Solexa) sequencing
50
reversible terminators
51
comparison of next generation sequencing platforms
52
Single molecule DNA Sequencing
53
Microcapillary-based SM DNA sequencing (M.Sauer et al. Heidelberg)
54
Microcapillary-based SM DNA sequencing
55
Microcapillary-based SM DNA sequencing
56
Flow-cytometry based DNA sequencing
57
cleavage vs control
58
DNA-sequencing: the Helicos-approach
step 1
59
DNA-sequencing: the Helicos-approach
step 2
60
DNA-sequencing: the Helicos-approach
step 3
61
DNA-sequencing: the Helicos-approach
step 4
62
DNA-sequencing: the Helicos-approach
step 5
63
DNA-sequencing: the Helicos-approach
step 6
64
DNA-sequencing: the Helicos-approach
step 7
65
DNA-sequencing: the Helicos-approach
step 8
66
DNA-sequencing: the Helicos-approach
step 9
67
helicos tool
68
correlogram
69
DNA target template
70
HELICOS & FRET
71
Pacific biosciences approach:
72
metal film
73
Pore-based sequencing
