Bio-Rad Model 491 Prep Cell and Mini Prep Cell - Manual (Page 24)

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Table 1. Recommended monomer concentrations.
Size Range %T Range
15–30 kD 6–10%
30–50 kD 9–12%
50–70 kD 7–10%
70–100 kD 5–9%
100-200 kD 4–8%
The procedure goes as follows:
1. Cast 3–4 polyacrylamide mini-slab gels in the range suggested in Table 1 (and Figure 10).
2. Load a sufficient amount of protein for detection by silver staining (~100 ng/lane). Load at least one lane
with 10 µl prestained high or low molecular weight standards and one lane with SDS-PAGE silver stain
standards (optional).
3. Choose one of the prestained molecular weight standards which migrates closest to the molecular
weight of the proteins of interest to monitor the gel run. Start elec trophoresis and continue until the
prestained marker protein has either run off the end of the gel or, where the proteins of in terest are
smaller than the marker, until the standard protein has reached a designated location near the end of the
gel.
4. When electrophoresis is complete, silver stain the gels and dry them.
5. Measure the distance between the protein bands of interest (protein to be purified and its nearest
contaminant) in each gel. (Net distance is measured; i.e., measure the distance between the bottom of
the upper band and the top of the lower band.)
6. Plot the distance between bands versus %T and determine optimal %T from the cusp (or breakpoint) of
the graph. This is the optimal %T for use in the preparative gel tube. Resolution de creases at either side
of optimal %T. Please see Section 4.7 for examples.
4.3 Gel Length Determination for Preparative SDS-PAGE
Gel length is used to increase the resolution between proteins. However, longer gels are accompanied by an
increase in band diffusion. The minimal gel length depends on the differ ence in molecular weight be tween the
protein of interest and its nearest con taminant. The size difference be tween these two proteins (∆MW) is in versely
related to the gel length. Small size differences require longer gels to produce the best resolu tion. The sample
load, i.e. the amount of the protein of interest and its nearest contaminant, also affects resolution. Resolution
can be improved by either decreasing the sample load or increasing the gel length. Refer to Table 2 to select
the gel length for optimizing resolution and recovery of the protein of in ter est.
4.4 Gel Tube Size Selection
Both the protein load and the molecular weight difference between the pro tein of interest and its nearest
contaminant must be considered when se lect ing the size of the gel tube. Two different gel tubes are provided:
a 28 mm (internal diameter) gel tube which forms a gel with a 3.6 cm
2 gel upper surface and a 37 mm (internal
diam eter) gel tube with an 8.2 cm 2 gel surface area. Resolution between proteins with very small molecular
weight differences will improve approximately 1.5-fold for identical protein loads when the 37 mm column is
used instead of the 28 mm. This increase in resolution is accompanied by a higher dilution factor due to the
larger volume elution chamber.