Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 24)

Mini-PROTEAN TGX
Any kD 4–20% 4–15% 12% 10% 7.5%
Broad Range Unstained
200
116
97.4
66
45
31
21.5
14.4
6.5
200
116
97.4
66
45
31
21.5
14.4
6.5
200
116
97.4
66
45
31
21.5
14.4
6.5
200
116
97.4
66
45
31
21.5
14.4
200
116
97.4
66
45
31
21.5
200
116
97.4
66
45
31
Any kD 4–20% 4–15% 12% 10% 7.5%
Precision Plus Protein Unstained
250
150
100
75
50
37
25
20
15
10
250
150
100
75
50
37
25
20
15
10
250
150
100
75
50
37
25
20
15
10
250
150
100
75
50
37
25
20
15
250
150
100
75
50
37
25
20
250
150
100
75
50
37
Mini-PROTEAN TGX
Any kD 4–20% 4–15% 12% 10% 7.5%
Broad Range Unstained
200
116
97.4
66
45
31
21.5
14.4
6.5
200
116
97.4
66
45
31
21.5
14.4
6.5
200
116
97.4
66
45
31
21.5
14.4
6.5
200
116
97.4
66
45
31
21.5
14.4
200
116
97.4
66
45
31
21.5
200
116
97.4
66
45
31
Any kD 4–20% 4–15% 12% 10% 7.5%
Precision Plus Protein Unstained
250
150
100
75
50
37
25
20
15
10
250
150
100
75
50
37
25
20
15
10
250
150
100
75
50
37
25
20
15
10
250
150
100
75
50
37
25
20
15
250
150
100
75
50
37
25
20
250
150
100
75
50
37
Fig. 4.2. Examples of migration charts. The protein standards
were run on Mini-PROTEAN
® TGX ™ gels.
Fig. 4.1. Separation of proteins by SDS-PAGE after separation by IEF. The IPG strip containing proteins already separated by pI is applied
to the top of a polyacrylamide gel. The proteins are then separated according to size (MW) by SDS-PAGE.
Low pH High pH
High MW
Low MW
Second Dimension
SDS-PAGE,
separation by MW
First Dimension
Isoelectric focusing (IEF),
separation by pl
44 45
2-D Electrophoresis Guide Theory and Product Selection Chapter 4: The Second Dimension: SDS-PAGE
Selection of Polyacrylamide Gels
Polyacrylamide gels are prepared by free radical
polymerization of acylamide and a comonomer
crosslinker such as bis-acrylamide. By convention,
gels are characterized by two parameters that
determine pore size: total monomer concentration
(%T, in g/100 ml) and weight percentage of crosslinker
(%C). SDS-PAGE gels typically have a %C of 2.7%,
and the %T is varied to give separation characteristics
appropriate to the experimental needs. %T determines
the relative pore size of the resulting polyacrylamide
gel, with higher %T resulting in smaller pores and
separation characteristics more appropriate for
smaller proteins.
Gels are either purchased as commercial precast
gels or cast in the laboratory using unpolymerized
monomer and buffer components. Precast gels are
available in smaller formats to fit commercially available
electrophoresis cells. These are appropriate for the
second dimension when the first dimension is run on
7 cm or 11 cm IPG strips. Larger second dimensions
are generally run on lab-cast gels.
Protein Separation by Size
The second-dimension separation is by protein size
(mass) using SDS-PAGE. The proteins separated in
IPG strips by IEF in the first dimension are applied to
polyacrylamide gels and separated a second time by
SDS-PAGE (Figure 4.1).
A two-step equilibration process prepares the proteins
for SDS-PAGE. The proteins are complexed with
SDS, reduced with DTT, and then alkylated with
iodoacetamide. Treatment of the proteins with SDS
yields protein-SDS complexes with a consistent
charge-to-mass ratio. When electrophoresed through
a polyacrylamide gel, these complexes migrate with a
mobility that is related logarithmically to mass. As the
proteins migrate through the gel, the pores of the gel
sieve proteins according to size.
Choice of Gel Percentage (Composition)
Gels for SDS-PAGE are made with either a single,
continuous %T throughout the gel (single-percentage
gels) or a gradient of %T (gradient gels). Gradient gels
are cast with acrylamide concentrations that increase
from top to bottom so that the pore size decreases
as proteins migrate into the gels. Single percentage
gels are cast in the laboratory by simply pouring the
appropriate percentage of acrylamide, along with
bis-acrylamide, buffer, initiator, and catalyst, into a
gel cassette prepared using glass plates and spacers
clamped together. The mixture is poured into the
cassette and allowed to polymerize. A stacking layer
is not necessary for second-dimension gels. Gradient
gels may also be cast in the laboratory using solutions
of differing acrylamide percentage and a gradient
maker. Typical gel compositions are 7.5–20% for
single-percentage gels, and 4–15% to 10–20% for
gradient gels.
Use protein migration charts and tables to select
the gel type that offers optimum separation of your
sample (Figure 4.2):
■ ■
Use single-percentage gels to separate bands of
similar size. Since optimum separation occurs in the
lower half of the gel, choose a percentage in which
the protein of interest migrates to the lower half of
the gel
■ ■
Use gradient gels to separate a broad range of
protein sizes. Gradient gels allow resolution of
both high- and low-molecular weight bands on the
same gel. The larger pore size towards the top of
the gel permits resolution of larger molecules, and
decreasing pore sizes toward the bottom of the gel
restrict excessive separation of small molecules.
Gradient gels are often the most appropriate choice
for 2-D electrophoresis, which is most often applied
to complex samples with proteins spanning a large
size range
■ ■
For new or unknown samples, use a broad gradient
gel (for example, 4–20 %T or 8–16 %T or Bio-Rad’s
Any kD
™ 3 formulation) for a global evaluation of the
sample, and then move to an appropriate single-
percentage gel for more detailed investigation of a
particular size range of interest
3 Bio-Rad’s Any kD formulation provides separation of 10–250 kD
proteins, with the best resolution in to the 20–100 kD range.
These gels are useful for screening samples or for 2-D applications
aimed at rapid protein analysis.