Bio-Rad Model 491 Prep Cell and Mini Prep Cell - Manual (Page 41)

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9.3 Discontinuous Native-PAGE
Acrylamide Concentration – Gel Pore Size
By convention, polyacrylamide gels are characterized by the figures (%T/%C), where %T is the weight
percentage of total monomer including crosslinker (in g/100 ml) and %C is the proportion of crosslinker as a
percentage of total monomer. For both the analytical gels and the preparative gels use 2.67% N, N’-methylene-
bis acry lamide crosslinker (premixed acrylamide:bis in the ra tio 37.5:1 can also be used). The total monomer
concentra tion determines the pore size of the gel and is re ferred to as %T.
The mobilities of native proteins can be modified by changing the pore size of the gel. This change in pore size
is accomplished by changing the amount of acry lamide (%T) in the gel. As the pore size changes, the change
in mobility differs for each protein in the sample mix. The result is that the separation pattern can be altered by
changing the acry lamide concentration.
The optimum gel concentration for preparative work is determined empiri cally as the one that best separates
the protein of interest from its contami nants. In native preparative PAGE, gels with large pores are preferred
be cause of the relatively high migration rates they allow. For discontinuous na tive PAGE in the Model 491 prep
cell, gels be tween 4% and 10%T/2.67%C provide op timal resolution. Gel lengths of 4–5 cm are sufficient to
resolve most pro teins. Determination of the optimum acry lamide concentration is accom plished using mini-slab
gels as follows.
Acrylamide Concentration – Optimization Procedure
Selection of the optimum gel concentration begins with running the sample in a series of analytical mini-gels
composed of 4%, 6%, 8%, and 10% acry lamide. When the marker dye reaches the bottoms of the gels, the
runs are terminated and the gels are stained. In each gel, the separation of the pro tein of interest relative to its
contaminants is assessed. The gel with the lowest acrylamide concentra tion that gives the best resolution is
the one to use for native preparative PAGE.
A graphical way to determine the optimum acrylamide concentration from these analytical gels is by comparing
the relative mobilities (Rf) of the protein of interest and its nearest contaminant. (To calculate Rf values see
Section 3.7.) To achieve this, plot the relative mobilities of the protein of interest and its nearest contami nant,
as measured in each gel, against acry lamide percentage. From this graph, choose the lowest acrylamide
con centration that gives the greatest difference in Rf be tween these two proteins. This is the %T to use for
preparative native-PAGE in the Model 491 prep cell.
Polyacrylamide Gel Recipes
A. Prepare a 30% Acrylamide Stock Solution
Acrylamide/bis (30%T/2.67%C)
Acrylamide - 146.0 g
N,N’-bis-methylene-acry lamide (Bis) - 4.0 g
Dissolve in 350 ml deionized water then adjust to 500 ml with deion ized wa ter. Filter and store at 4°C in the
dark.
(Preweighed Acrylamide/bis 37.5:1 mixture or 30% acrylamide/bis stock so lu tion can be substituted.
Caution: Acrylamide monomer is a neurotoxin.
Avoid breathing acrylamide dust, do not pipet acrylamide solutions by mouth, and wear gloves when handling
acrylamide powder or solutions con taining it. For dis posal of unused acrylamide, add bis-acrylamide to create
a gel, induce polymer ization, and discard the solidified gel.
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