Bio-Rad Model 491 Prep Cell and Mini Prep Cell - Manual (Page 22)

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Section 4
Optimizing Running Conditions for Preparative SDS-PAGE
The Model 491 prep cell is designed for separating a single component from its nearest con taminant. The
conditions required to achieve optimum resolution may be different than those of analytical electrophoresis. In
analytical SDS-PAGE, optimum reso lu tion is needed over a wide molecular weight range, whereas in
preparative SDS-PAGE the protein of interest needs to be separated from its nearest contaminant after
traveling the entire length of the gel.
The three variables to be considered for optimum resolution in preparative SDS-PAGE are:
4.1 Gel pore size
4.3 Gel length
4.4 Gel tube diameter
4.1 Gel Pore Size (Determining Optimum %T)
Since each protein purification is unique, it is important to first optimize the run ning conditions for each
application on an analytical level prior to prepara tive fractionation. In general, the best purification will be
achieved if the sample is at least partially purified prior to a preparative run. Any op timization procedure
should be carried out using the same protein sample as will be ap plied to the Model 491 prep cell.
The most important parameter in preparative SDS-PAGE is the pore size of the gel. The gel pore size is a
function of the acrylamide monomer concentra tion (%T) used to cast a gel. The monomer concentration
which best re solves two protein bands will vary de pending on the molecular weights of the pro teins of
interest. Changing the gel com position from the optimal concentration, by increasing or decreasing the
monomer concentra tion, will ultimately decrease reso lution.
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 for optimal resolution is re ferred to as optimal %T.
The pore size providing the optimum resolution for most proteins is that which results in a relative mobility (Rf)
value be tween 0.55–0.6. Optimal reso lu tion is not achieved with monomer concentra tions other than the
optimum value.
Rf values for specific proteins are obtained from the mini-gels that were run to op timize conditions for the
Model 491 prep cell. To calculate Rf values for specific proteins use this formula:
Distance that the protein of interest migrated
Distance that the tracking dye migrated
The Rf value obtained from a mini gel can be used to estimate when a protein will elute from the Model 491
prep cell when the same concentra tion of acrylamide is used in both the mini gel and the preparative gel.
For those samples in which the molecular weight difference between the pro tein of interest and its nearest
contaminant is ≥10%, the optimum %T can be selected from Figure 10 in the following section. For those
samples in which the molecular weight difference be tween the protein of interest and its nearest contaminant is
≤10%, consult Section 4.3.
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Rf =