long question (10)

Question 1

compare two techniques used in proteomics

Question 2

name three techniques used in proteomics

Answer 2

• two-dimensional gel electrophoresis
• two-dimensional difference gel electrophoresis
• mass spectrometry

Question 3

what is two-dimensional gel electrophoresis

Answer 3

• used for detection & analysis of complex protein mixture

Question 4

2DE steps

Answer 4

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

Question 5

2DE advantages

Answer 5

• resolves hundred of proteins
• identifying new proteins
• measures their relative abundance

Question 6

2DE disadvantages

Answer 6

• challenging spot-to-spot comparison/matching
• proteins do not migrate to the same point on each gel
• need for computer-based spot matching

Question 7

two-dimensional difference gel electrophoresis

Answer 7

• allows one to compare two or three protein samples simultaneously on the same gel

Question 8

2D-DIGE steps

Answer 8

• 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

Question 9

2D-DIGE advantages

Answer 9

• 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

Question 10

2D-DIGE disadvantages

Answer 10

• ambiguity for lower abundance protein spots
• dye-labelling
• relatively expensive
• lesser abundance proteins mostly unidentifiable