1 Flashcards
How are large quantities of the protein of interest made?
- insert cDNA for gene of interest into expression vector using restriction enzymes
- expression vectors use an inducible bacteriophage promoter to drive high levels of gene expression
- induce gene expression (and thus protein production) using temperature shift or adding chemicals
- harvest the dead bacteria in a centrifuge
- lyse bacteria using detergent
- purify protein
Epitope tagging system
- used to visualise proteins when no antibodies are available
1. DNA sequence for epitope tag is fused in frame to the cDNA during cloning
2. protein of interest recombinant with epitope tag
Epitope tag
Peptide which binds to its antibodies
DEFINE: Epitope
Small regions of proteins with very high specificity that are recognised by antibodies
How to create protein-specific antibodies?
- clone cDNA and fuse DNA sequence for epitope tag to the cDNA
- make a lot of the protein of interest
- purify protein using antibody-affinity purification - antibodies on the beads bind to epitope tag
- inject protein into rabbits over a 3 month period - rabbits produce antibodies against protein of interest
- purify antibodies from serum
What colour substrate does alkaline phosphatase produce?
Blue
What colour substrate does horse radish peroxidase produce?
Brown
Secondary antibodies
many secondary antibodies carrying tag bind to one primary antibody - amplifies signal
Atomic mechanism of GFP
- GFP absorbs high energy light = 475nm (shorter wavelength)
- electron shifted out of normal orbit to a unstable, higher energy state. this is temporary.
- electron moves back into normal orbit and loses energy in the form of light. electron emits light at a longer wavelength = 510nm
What does gene fusion consist of?
deleting stop codon and fusing GFP to C terminus and then introducing new stop codon
What should a GFP reporter construct be used for?
To mark specific cells so they can be distinguished from their neighbours
