long question (10) Flashcards
1
Q
compare two techniques used in proteomics
A
2
Q
name three techniques used in proteomics
A
- two-dimensional gel electrophoresis
- two-dimensional difference gel electrophoresis
- mass spectrometry
3
Q
what is two-dimensional gel electrophoresis
A
- used for detection & analysis of complex protein mixture
4
Q
2DE steps
A
first dimension
- separates proteins according to the isoelectric point
- their net charge is zero compared to the pH of the gel
second dimension
- tub gel is rotated 90 degrees
- separates proteins according to molecular weight
5
Q
2DE advantages
A
- resolves hundred of proteins
- identifying new proteins
- measures their relative abundance
6
Q
2DE disadvantages
A
- challenging spot-to-spot comparison/matching
- proteins do not migrate to the same point on each gel
- need for computer-based spot matching
7
Q
two-dimensional difference gel electrophoresis
A
- allows one to compare two or three protein samples simultaneously on the same gel
8
Q
2D-DIGE steps
A
- label protein extracts with fluorescent dyes (CyDyes)
- approx. one dye molecule per protein
- separated on the same gel
- labelled proteins migrate simultaenously on the 2-DE gel & have distinct excitations
- image gel with CyDye (CyTM2, Cy3 qnd Cy5) specific scanner
9
Q
2D-DIGE advantages
A
- avoids gel-to-gel variability
- simplified spot matching & quantitation
- less gels to match when multiplexing
- comparison allows detection of changes in protein expression levels & post-translational modifications
10
Q
2D-DIGE disadvantages
A
- ambiguity for lower abundance protein spots
- dye-labelling
- relatively expensive
- lesser abundance proteins mostly unidentifiable