6.2 Cell Biology Methods II

Question 1

SDS PAGE

Answer 1

Very common method of separating proteins by electrophoresis. Motility is a function of length, conformation and charge of the molecule

Question 2

What does SDS stand for?

Answer 2

Sodium dodecyl sulfate = an anionic detergent

Question 3

Isoelectric focusing

Answer 3

pH gradient set up, + charge at the top, - charge at the bottom. Proteins travel towards the center. At the isoelectric point, the protein has no net charge and no longer migrates

Question 4

2D gel

Answer 4

SDS migration on the Y axis, Isoelectric on the x axis. Advantages = molecules are more effectively separated in 2D rather than 1D electrophoresis…..it is unlikely that 2 molecules will be similar in 2 distinct ways

Question 5

Western Blotting (immunoblotting)

Answer 5

Separated protein bands are transferred to a carrier membrane = Blotting. Proteins adhere to the membrane in the same pattern. Can then be accessible for antibody binding for detection.

Question 6

ELISA

Answer 6

only 1 protein can be detected

Question 7

Epitope tag

Answer 7

short peptide sequences recognized by a monoclonal antibody.

Epitope tagging is used to concentrate or purify a protein.

Question 8

What are some of the methods used to study the function of a protein?

Answer 8

• gene replacement
• gene knockout
• gene addition

Question 9

How are transgenic plants/animals produced?

Answer 9

Random incorporation of foreign gene into a mouse genome/leaf disc incubated with genetically modified argrobacterium

Question 10

What methods can be used to determine the structure of proteins?

Answer 10

• X-ray crystallography

- NMR spectroscopy

Question 11

What is mass spectrometry?

Answer 11

Ionizes chemical species and sorts the ions based on their mass-to-charge ratio —-> measures the masses within a sample