VRAGEN CONCEPTS ORIGINEEL EXAMEN 1E
ZIT
VRAAG 9 = IS GESCHRAPT DUS MOETEN WE NIET KENNEN!!!!
Voor het inhaalexamen van mijn jaar 1e zit (1e Ma, 1e semester,
2022-2023) Kwamen er heel wa vragen terug van die voorbeeld
examenvragen da hij er zelf had opgezet op BB maar net voor
het inhaalexamen heeft hij dit er af gedaan en het staat er nog altijd
niet op dus mss voor 2de zit de moeite om is te bezien & ook
nog vragen uitwerken!!!
HOOFD VRAGEN:
1) Vraag 1: 2D-DIGE: Principle, advantages/disadvantges, sample prep (8p):
Concept uitleggen + Advantages en Disadvantages geven & Sample prep een Protocol (zowa sample prep
en van heel het proces alle stappen doen denk) ervoor uitwerken:
Antwoord: CONCEPT
,Differential in gel electrophoresis (2D-DIGE): It involves 2D-GE separation of pre-labeled
fluorescent samples. Three dyes are imployed: Control (internal standard, Cy2), Cy3 and Cy5.
They all have the same mass so they don’t influence the separation and they have the same
hydrophobicity. By looking at a different wavelength you can see the different dyes:
differential fluorescent scanning generates a quantitative multiplexed output. Cy dye
fluorophore containing an NHS ester it covalently binds to Lys of target protein via an
amide link. Trypsine digestion can be used afterwards because you won’t be adding the dyes
in a high amount. The cy2 image will be identical in both samples because it contains a bit of
both samples. It’s also a normalization tool. You can compensate the signal based on the cy2
image. Now you can run 3 samples on one gel: 2 you want to quantify and 1 internal
standard, instead of making separate gels.
Some dyes prefer certain proteins so you should also do a reverse dye: switching all labels so
you avoid mistakes. In the analysis:
Yellow spots: intensities of red & green were roughly the same
Red spots: high signal red, low signal green differential spot: upregulated in sample with red dye
Green spots: high signal green, low signal red differential spot: upregulated in sample with green
dye
Take home points:
DIGE Involves 2D GE separation of pre-labeled fluorescent samples
Three dyes employed Control (Cy2, internal standard), Cy3, Cy5
, Cy2, Cy3 and Cy5 labeled samples are multiplexed and co-run on 2D GE
Differential fluorescent scanning generates a quantitative multiplexed 2D GE output
Cy dye fluorophore containing an NHS ester it covalently binds to Lys of target
protein via an amide link.
Advantages en Disadvantages:
Advantages:
1) Good for hydrophobic protein analysis & post-translational analysis
2) It overcomes limitations in traditional 2D electrophoresis that are due to inter-gel
variation. This can be considerable even with identical samples. Since the proteins
from the different sample types (e.g. healthy/diseased, virulent/non-virulent) are run
on the same gel they can be directly compared. To do this with traditional 2D
electrophoresis requires large numbers of time-consuming repeats.
3) In experiments comprising several gels, a common technique is to include an internal
standard in each gel. The internal standard is prepared by mixing together several or
all of the samples in the experiment. This allows the measurement of the abundance
of a protein in each sample relative to the internal standard. Since the amounts of
each protein in the internal standard is known to be the same in every gel, this
method reduces inter-gel variation.
Disadvantages:
Je moet die Cy2, Cy3 en Cy5 toevoegen als extra stap. Dat hoef je bij gewone 2D-GE page
niet te doen Dus dat is meer sample manipulation compared to simple 2D page
ZIT
VRAAG 9 = IS GESCHRAPT DUS MOETEN WE NIET KENNEN!!!!
Voor het inhaalexamen van mijn jaar 1e zit (1e Ma, 1e semester,
2022-2023) Kwamen er heel wa vragen terug van die voorbeeld
examenvragen da hij er zelf had opgezet op BB maar net voor
het inhaalexamen heeft hij dit er af gedaan en het staat er nog altijd
niet op dus mss voor 2de zit de moeite om is te bezien & ook
nog vragen uitwerken!!!
HOOFD VRAGEN:
1) Vraag 1: 2D-DIGE: Principle, advantages/disadvantges, sample prep (8p):
Concept uitleggen + Advantages en Disadvantages geven & Sample prep een Protocol (zowa sample prep
en van heel het proces alle stappen doen denk) ervoor uitwerken:
Antwoord: CONCEPT
,Differential in gel electrophoresis (2D-DIGE): It involves 2D-GE separation of pre-labeled
fluorescent samples. Three dyes are imployed: Control (internal standard, Cy2), Cy3 and Cy5.
They all have the same mass so they don’t influence the separation and they have the same
hydrophobicity. By looking at a different wavelength you can see the different dyes:
differential fluorescent scanning generates a quantitative multiplexed output. Cy dye
fluorophore containing an NHS ester it covalently binds to Lys of target protein via an
amide link. Trypsine digestion can be used afterwards because you won’t be adding the dyes
in a high amount. The cy2 image will be identical in both samples because it contains a bit of
both samples. It’s also a normalization tool. You can compensate the signal based on the cy2
image. Now you can run 3 samples on one gel: 2 you want to quantify and 1 internal
standard, instead of making separate gels.
Some dyes prefer certain proteins so you should also do a reverse dye: switching all labels so
you avoid mistakes. In the analysis:
Yellow spots: intensities of red & green were roughly the same
Red spots: high signal red, low signal green differential spot: upregulated in sample with red dye
Green spots: high signal green, low signal red differential spot: upregulated in sample with green
dye
Take home points:
DIGE Involves 2D GE separation of pre-labeled fluorescent samples
Three dyes employed Control (Cy2, internal standard), Cy3, Cy5
, Cy2, Cy3 and Cy5 labeled samples are multiplexed and co-run on 2D GE
Differential fluorescent scanning generates a quantitative multiplexed 2D GE output
Cy dye fluorophore containing an NHS ester it covalently binds to Lys of target
protein via an amide link.
Advantages en Disadvantages:
Advantages:
1) Good for hydrophobic protein analysis & post-translational analysis
2) It overcomes limitations in traditional 2D electrophoresis that are due to inter-gel
variation. This can be considerable even with identical samples. Since the proteins
from the different sample types (e.g. healthy/diseased, virulent/non-virulent) are run
on the same gel they can be directly compared. To do this with traditional 2D
electrophoresis requires large numbers of time-consuming repeats.
3) In experiments comprising several gels, a common technique is to include an internal
standard in each gel. The internal standard is prepared by mixing together several or
all of the samples in the experiment. This allows the measurement of the abundance
of a protein in each sample relative to the internal standard. Since the amounts of
each protein in the internal standard is known to be the same in every gel, this
method reduces inter-gel variation.
Disadvantages:
Je moet die Cy2, Cy3 en Cy5 toevoegen als extra stap. Dat hoef je bij gewone 2D-GE page
niet te doen Dus dat is meer sample manipulation compared to simple 2D page
