Bio-Rad Bio-Gel P Polyacrylamide Gel - Manual (Page 6)

5. Allow entire sample to enter the column and discard
the first 3.0 ml of effluent.
6a. Add 4.0 ml of buffer to elute the higher molecular
weight component(s), while collecting the 4.0 ml
fraction from the column. For a more precise
collection method refer to step 6b.
6b. Elute the desired components with 8 ml of buffer,
while collecting 1 ml fractions from the column.
Analyze the fractions with the appropriate detector
and plot the results against the fraction number. Pool
the desired fractions (See Figure 5.1).
Minimal Dilution Protocol
1. Remove the upper cap, and pour off the excess
buffer above the top frit.
2. Add 20 ml of the appropriate buffer to the column
(fill to the 30 ml mark), and snap off the bottom tip
to start the column flowing.
3. Allow the buffer to drain to the top frit. The column
will not run dry. Flow will stop when the buffer
level reaches the top frit.
4. Measure sample volume, then add sample to the
column.
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5.1 Desalting or Buffer Exchange
Protocols
The following desalting protocols are recommended
for sample volumes ranging from 100 µl to 3.0 ml. For
volumes < 100 µl use Bio-Spin chromatography
columns. For volumes > 3.0 ml use Bio-Gel P-6DG gel
packed into an appropriate column.
General Protocol
1. Remove the upper cap, and pour off the excess
buffer above the top frit.
2. Add 20 ml of the appropriate buffer to the column
(fill to the 30 ml mark), and snap off the bottom tip
to start the column flowing.
3. Allow the buffer to drain to the top frit. The column
will not run dry. Flow will stop when the buffer
level reaches the top frit.
4. Add a 3.0 ml sample to the column. If sample is less
than 3.0 ml add buffer to reach a total volume of 3.0
ml or perform minimal dilution protocol to minimize
sample dilution.
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