Bio-Rad Bio-Gel P Polyacrylamide Gel - Manual (Page 8)

Fig. 5.2. Separation of radiolabeled protein
from unincorporated radiolabel.
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5.2 Application 1: Separation of
Incorporated Radiolabeled Proteins
from Unincorporated Radiolabel
1. Follow steps 1 through 3 in the general desalting
procedure, Section 5.1.
2. Apply the radiolabeled protein solution to the
column, allowing the sample to run completely into
the column. (For best results, the sample volume
should be between 1.0 and 3.0 ml.)
3. Elute with 10 ml of buffer, collecting 1.0 ml
fractions.
4. Count 10 µl of each fraction in an appropriate
scintillation cocktail.
5. Plot CPM against fraction number to determine
which fractions to pool (see Figure 5.2).
6. Discard the column and unpooled fractions in an
appropriate radioactive waste container.
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Conditions
Column:
Econo-Pac 10 DG desalting column
Sample: BSA in 250 mM NaCI
Buffer: H
2
O
Flow rate: 1.0 ml/min
Peaks:
1. 125 l labeled FSH
2. 125 l (unincorporated)
CPM
Fraction number
LIT81D 7/9/98 8:37 AM Page 10