Bio-Rad Model 491 Prep Cell and Mini Prep Cell - Manual (Page 21)

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3.7 Loading the Sample
Care fully load the sample on the surface of the gel through the sample load ing guide with the sample
application syringe. Layer the sample under the electrophore sis buffer above the gel. Make sure the
stacking gel is not punc tured with the PTFE tubing.
Once the sample is loaded, place the lid on the cell and attach the cables to the power supply. Set the
power supply to the appropriate setting and begin elec trophoresis.
3.8 Elution Rate, Detection, Collection, and Analysis
Elution Rate
For the highest yield of purified protein we recommend an elution buffer flow rate of 0.75–1.0 ml/min.
Fractions of 2.5 ml usually provide sufficient separation. Fraction collec tion should begin after the ion/dye
front has eluted. For preparative SDS-PAGE, Table 5 shows the approximate elution times of purified
proteins for gels run with the opti mum %T and proper power condi tions. Refer to Section 9 for native-
PAGE.
Detection
Elution of molecules can be monitored with an ultraviolet detector and chart recorder. However, with
preparative PAGE, a UV monitor does not usually provide an adequate representation of the fractionation.
The elution profile on the chart recorder tracing cannot replace electrophoretic analysis of indi vidual fractions
for determining the distribution of proteins and the level of contamination in each fraction. The ion front will
be seen on the UV chart recording as a high-absorbance peak or it will be in the fractions containing the
sample-buffer dye. Individual peaks can sometimes be seen on a chart recording, but in many instances the
sample will yield a broad, poorly defined trace registering slightly above baseline. See Figure 13 for an
example of an elution profile for a starting sample containing only three pro tein bands.
Collection and Analysis
To determine which fractions contain the protein of interest, individual prep cell frac tions must be analyzed
by slab gel electrophoresis. It is recommended that an analysis be performed by running every fifth or tenth
fraction past the ion front (i.e., fractions collected every 15 to 30 minutes) in mini gels. When the region with
the protein of interest is identified, every fraction within that region should be analyzed to determine the level
of contamination.
Fractions containing the puri fied protein can be pooled and concentrated as required for further analysis.
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