Protein Protocols Protein Protocols

Purification and Assessment of Quality 981
Fig. 1. Gel filtration of radioiodinated kallikrein of rat submandibular gland. The pure
enzyme (10 µg) was iodinated with 125 I (18.5 MBq) by the chloramine T method. It was then
purified on a column (1 × 20 cm) of Sephadex G-75 resin at a flow rate of 20 mL/h and
collecting fractions of 0.6 mL. Aliquots (10 µL) of each fraction were measured for radioactivity.
By radioimmunoassay, immunoreactive protein was found only in the first peak, and
more than 90% of that radioactivity was bound by the antiserum to kallikrein from rat submandibular
gland.
4. Polyethylene glycol produces precipitation of antibody and antibody-bound protein with
little precipitation of unbound protein. Potassium iodide decreases the precipitation by
polyethylene glycol of unbound protein (3).
5. A typical example of the calculation of yield of iodination and specific radioactivity is
as follows:
Counts of 10 µL incubation mixture prior to gel filtration = 1,567,925 counts/10 s (1)
Some of the radioiodinated protein is likely to bind to the reaction vessel and other
surfaces, so it is best to calculate the radioactivity associated with the labeled protein in
terms of the difference between total radioactivity applied to the column and the radioactivity
associated with the unreacted iodide.
Counts in 10-µL aliquots of iodide peak = 388,845 counts/10 s (2)
Counts in 10-µL aliquots of protein peak
= 1,567,925–388,845 counts/10 s = 1,179,080 counts/10 s (3)
Yield of radioiodination reaction = % incorporation of 125 I into protein
(1,179,080 × 100)/1,567,925 = 75.2% (4)