Biomedical Engineering Reference
In-Depth Information
µ
l of Soln. A (4 - 7 MBq) into a 4-ml polystyrene or
siliconized glass test tube. Then add 25
Pipet 5 - 10
µ
µ
l Soln. B, followed by 10
l
µ
µ
of protein solution (2 - 5
g, dissolved in Soln. C), 10
l Soln. D,
µ
and 100
l Soln. E. Vortex after each addition. Fill up to 1 ml with
Soln. G and count for total radioactivity.
Equilibrate a 1
10 cm Sephadex G-25 column with 50 ml
Soln. F. Apply the iodinated sample, elute with Soln. F, and collect
1-ml fractions. The iodinated protein appears in the void volume,
and free iodine-125 elutes with the total volume. Estimate its ra-
dioactivity and calculate the specific radioactivity (Bequerel per
milligram of protein).
If the iodinated protein will be used in bioassays, check for
biologic activity (antigenicity, enzymic activity, ligand binding,
etc.) in comparison with the not-labeled starting material.
×
6.4.2 Iodination with B
OLTON
-H
UNTER
Reagent
Bolton-Hunter reagent (N-succinimidyl 3(4-hydroxy-5-[
125
I]-
Solutions/Reagents
A
/
iodophenyl)propionate) (70 - 150 TBq
mMol) in benzene
B .1M borate buffer, pH 8.5
C .2M glycine in Soln. B
D
0.1% serumalbumin (w/v) or 0.1% gelatin (w/v) in 50 mM
phosphate buffer, pH 7.5
Pipet about 5 - 10 MBq (about 250 000 - 500 000 dpm) of Soln. A into
a 4-ml test tube. Carefully vaporize the solvent by a gentle stream
of dry nitrogen. Add 10
µ
µ
g
of protein in Soln. B, and agitate the tube in an ice bath for 30 min.
Add 0.5 ml Soln. C and continue shaking at 0
◦
C.Fillupto1ml
with Soln. D after an additional 5 min. Separate the labeled protein
from the unreacted reagent as described in Protocol 6.4.1.
l of protein solution, containing 2 - 5
References
Bolton AE (1985) Radioiodination techniques (Amersham Review 18), 2nd
ed., Amersham International plc, Amersham
Bolton AE, Hunter WM (1973) Biochem J 133:529
Hermanson GT (1996) Bioconjugate techniques. Academic Press, New
York, p 414
6.5 Scintillation Cocktails
for Liquid Scintillation Counting
The scintillation cocktail chosen depends on the radioisotope and
the type and amount of solvent containing the labeled compound.
High-energy
β
emitting isotopes, such as 32-phophorus, are de-
tectable by several methods such as liquid scintillation (Cerenkov
