Labelling and Radiolabels Flashcards
List FIVE different types of label that can be added to proteins and/or DNA
- Flourescent
- Radioactive
List the merits and limitations of radioactive labels
MERITS
* Small size
* High sensitivity
* Well-developed chemistry and instrumentation
* High signal to noise ratio
* High accuracy
LIMITATIONS
* Biological hazard
* Decaying activity
* Radioactive damage of sample
* Requires separation steps
What is alpha radiation?
Two protons and two neutrons bound together
What is beta radiation?
High energy electron
What is gamma radiation?
High energy, electromagnetic photon
What type of radiation is emitted by tritium 3H and 32 phosphorus 32P?
Beta radiation
What is the half life of 3H?
12.3 years
What is the half life of 32P?
14.3 days
Explain how proteins can be labelled with 35S
- S35 Methionine included as precursor in Master Mix
- PCR fragment incorporated into plasmid DNA
- Plasmid DNA transcribed into mRNA
- mRNA translated into protein with S35 at every Methionine position
What is the unit of radioactivity?
Original - Curie (Ci)
New - Becquerel (Bq)
What is a Bequerel?
1 radioactive decay per second
What is a Gray?
1 joule of energy absorbed by 1kg of tissue
What is a Sievert?
Absorbed dose weighted for harmfulness of different radiations
What is a Geiger-Muller counter?
Detects high-energy particles
What is liquid scintillation counting?
A method to quantify the radioactivity of low energy radioisotopes (mostly alpha and beta)
Explain the principle of liquid scintillation counting
- Aromatic solvent molecules excited
- Energy is transferred to scintillator (phosphor or fluor)
- Absorbed energy through scintillator produces excited states of the electrons, which decay to ground state and produce light pulse
- Light is detected by photomultiplier tube (PMT)
