Radioactivity in biology
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Radioactivity in the life sciences is used as a radiolabel, these in some applications have been substituted by fluorescent dyes. These radionuclei are synthesised in particle accelerators and have short half lives, giving them high maxium theoretical specific activity, lowering the time that is needed for them to be detected, compared to radionuclei with longer half lives like carbon-14.
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[edit] Radionuclei used
- Tritium: This very low energy emittor is used to label proteins, nucleic acids, drugs and toxins, but requires a tritium specific film or a tritium specific phosphor screen, and in a LSA the efficiency is 20-50% depending on the scintillation cocktail used. The maxium theorectical specific activity of tritium is 28.8 Ci/mmole (often though there are more than one tritium atom per molecule, for example tritiated UTP is sold by most suppliers with labelled 5 and 6 carbons).
- Carbon-14: not used very often as a radiolabel due to its long half life. maximum specific activity is 0.0624Ci/mmole. It is instead used a lot in other applications, such as radiometric dating or drug tests.
- Sulphur-35: used to label protein and nucleic acids. cysteine is an aminoacid with a thiol group. Nucleotides do not contain a sulphur group, the oxygen on one of the phosphate groups can be substituted with a sulphur, this thiophosphate acts the same as a normal phosphate group (although there is a slight bias against it by most polymerases). Maximum SA: 1494 Ci/mmole.
- Phosphorus-33: used to label nucleotides, it is less energetic than P-32, giving a better resolution. 5118 Ci/mmole. The disadvantage is its higher cost compared to P-32, as most of the bombarded P-31 will have acquired only one neutron and some 2 or more. 5118 Ci/mmole.
- Phosphorus-32: choice radionuclei for nucleotides, its high energy and low half life result in lower autoradiography exposure times. Unfortunatelly its higher energy requires acrylic glass protection and a film badge. 9131 Ci/mmole.
- Iodine-125: used for labelling tyroxine. Due to the fact that it can become airborn and the iodine is absorbed heavier precautions must be taken. 2176 Ci/mmole.
H3 is very low energy emittor, its waste disposal regulation depends on the institution, in some places it is acceptable to dispose of down the drain. C14, S35, P33 have similar emission energies. P-32 and I-125 are higher energy emitters. In a scintillation counter H-3 energy range window is between channel 5-360, C-14, S-35, P-33 and are in the window 361-660. P-32: 661 - 1024.
[edit] Detection
[edit] Quantitative
LSA, (Liquid scintillation assay or Liquid scintillation counting): a small aliquot or a filter or a swab is added to scintillation fluid and the plate or vial counter in a scintillation counter. Geiger counter: a quick and rough approximation of the activity can be obtained, tritium is not be detected.
[edit] Qualitative
Autoradiography: a membrane such as a northern blot or a hybridised slot blot) is put against a film that is then delevoped. Phosphor storage screen: the membrane is placed against a phosphor storage screen which is then scanned in a phosphorimager. This is 10 times faster and more precise than film and the result is already in digital form.
[edit] Microscopy
Electron microscopy: The sample is not exposed to a beam of electrons but detectors picks up the expelled electrons from the radionuclei. micro autoradiography imager: a slide is put against slintillation paper and in a PMT. When 2 diffenent radiolabels are used the computer calculates discriminates the two.
[edit] Methods
for radioligand binding assays see Schild regression DNA labelling leaving the nucleic acid intact (5' and 3' labelling)
[edit] Concentrations and Calculations
Taking as an example γ32P ATP, from the catalogues of the two major suppliers, Perkin elmer NEG502H500UC [1] or GE AA0068-500UCI [2]. A vial of label has a total activity, in this case 500μCi (other typical numbers are 250μCi or 1mCi). This is in a certain volume depending on the radioactive concentration, such as 5 or 10 mCi/mL: 50 or 25 μL. Not all molecules in the solution have a P-32 on the last phosphate: the specific activity gives the radioactivity concentration and depends on the radionuclei's half life. If every molecule were labeled you obtain the maximum theoretical specific activity that for P-32 is 9131 Ci/mmole. Due to precalibration and efficiency issues this number is never seen on a label. The values often found are 800, 3000 and 6000Ci/mmole, with this number it is possible to calculate the total chemical concentration and the hot to cold ratio. Calibration date is the date in which the vials activity is the same as on the label. Precalibration is the when the activity is calibrated in a future date to compansate for the decay occurred during shipping.
[edit] Safety
Radionuclei used in a lab are extremely faint compared to well known radioactive samples such as uranium. Nevertheless the effects of low doses are mostly unknown so many regulations exist to avoid unnecessary risks, such as skin or internal exposure. Due to the low penetration power and many variables invlved it is hard to convert a radioactive concentration to a dose. 1μCi of Phosphorus 32 on a square centimetre of skin (through a dead layer = 70μm thick) gives 7961 rad per hour. Similarly a mammogram gives an exposure of 300mrem on a larger volume (the US the average annual dose is 360 mrem)
