L21 Antibodies in cell signalling pt 4

Question 1

How can the phospho-protein content of a cell be analysed?

Answer 1

Phospho-protein array.

Question 2

What is a phospho-protein array?

Answer 2

Array for simultaneous detection of many phospho-proteins. Can be used to analyse different phosphorylations in the same protein too.

Question 3

What does flow cytometry allow in studying cell signaling?

Answer 3

Quantitative analysis of protein phosphorylation.

Allows small sub-populations of cells to be studied in complex samples (tissues).

Question 4

What are the practical considerations when studying protein phosphorylation?

Answer 4

Protease and phosphatase activity (must use inhibitors)

Protein loading (varying amounts)

Kinase activation may be independent of the phospho-specific site you are monitoring

Question 5

How are kinases and phosphatases accounted for when studying phospho-proteins?

Answer 5

Using inhibitors:

• Sodium fluoride : serine/threonine phosphoatase inhibitor.
• Sodium orthovanadate : tyrosine phosphoatase inhibitor

Question 6

What is ubiquitin?

Answer 6

Protein, PTM that is added to proteins. Direct their target for degradation.

Question 7

How are antibodies used to detect ubiquitination?

Answer 7

Detect gross changes in protein ubi.

Monitor ubi. of specific proteins.

Detect specific ubi. chains (K48, K63, K11 etc)

Question 8

What are the two degradative pathways?

Answer 8

Ubiquitin proteosome system

Autophagy-lysosome system.

Question 9

What are the three autophagy pathways?

Answer 9

Microatuophagy.

Chaperone-mediated autophagy.

Macroautophagy.