Yeast Lecture 3 Flashcards
What is a YAC?
Yeast artificial chromosome.
Enables cloning of big DNA fragments
Used for genomic maps and libraries
When is autophagy induced?
Under starvation conditions
What are the functions of intracellular proteolysis?
Recycling AA
Protein Quality control
Selective protein degradation
activation of proteins
What is the pulse chase assay?
- Radioactive labeling (PULSE)
Growth in presence of radioactive AA - Addition of excess cold AA (CHASE)
- Cell extract + antibody aufreinigung
- Gel electrophoresis and autoradiography
What does Pulse chase reveal
Fate of New Born proteins
What is Cycloheximide chase assay?
- Add Cycloheximide at point 0
(blocks translation and thus de novo synthesis) - Take samples
- Prepare cell extracts
- Gel electrophoresis and Western blot
What are advantages of Cycloheximide chase?
Starts from stady state level but only works if degradation system does not require de novo synthesis of proteins
What is Promotor shutoff assay?
- Change media at time point 0
(represses promotor) - Take samples
- Cell estract
- Gel electrophoresis and Western blot
What are advantages of Promotor shutoff assay?
Starts from steady state level but delayed effect and artificial expression level
What is the Aptamer method?
Selectively shuts down translation of specific mRNA after addition of Tetracyclin then same as cycloheximide chase
What are advantages of Aptamers?
Starts at steady state level, fast and without delay but artificial expression level
What is tandem fluorescent protein timer?
Detects stability of proteins in vivo
Uses fusion protein. X-GFP-mCherry
Stable proteins have same expression GFP + mCherry
short lived protein much higher GFP
What are advantages of fluorescent protein timer?
High troughput protein stability assay at Steady state
What is SILAC
Stable isotope labeling by Amino acids in cell culture
Light medium and heavy medium -> analysis by mass spec
Whats special about YACs
Have own Telomeres and Centromeres
