Analytical Biochemistry 509 (2016) 12e14

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Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio

Technical note

A method for easily customizable gradient gel electrophoresis

Andrew J. Miller, Brandon Roman, Eric Norstrom*
Department of Biological Sciences, DePaul University, 2325 N. Clifton Ave, Chicago, IL 60614, USA

a r t i c l e i n f o a b s t r a c t

Article history: Gradient polyacrylamide gel electrophoresis is a powerful tool for the resolution of polypeptides by
Received 18 March 2016 relative mobility. Here, we present a simplified method for generating polyacrylamide gradient gels for
Received in revised form routine analysis without the need for specialized mixing equipment. The method allows for easily
1 July 2016
customizable gradients which can be optimized for specific polypeptide resolution requirements.
Accepted 4 July 2016
Available online 7 July 2016
Moreover, the method eliminates the possibility of buffer cross contamination in mixing equipment, and
the time and resources saved with this method in place of traditional gradient mixing, or the purchase of
pre-cast gels, are noteworthy given the frequency with which many labs use gradient gel SDS-PAGE.
Keywords:
Gradient
© 2016 Elsevier Inc. All rights reserved.
Gel
Electrophoresis
Polyacrylamide
SDS-PAGE
Glycine
Tricine

1. Article text
The separation of proteins based on their electrophoretic
properties using one dimensional polyacrylamide gel electropho-
resis (PAGE) and downstream analyses (e.g. Western Blot, staining,
protein excision and manipulation, etc.) have long been essential
tools in biochemistry [1,2]. While the acrylamide concentration can
be altered to produce different migration patterns, electrophoresis
with a gel of fixed concentration offers optimal resolution and
resolving power within a limited range of polypeptide masses [3].
Therefore, it is often desirable to use gels with an acrylamide
concentration gradient. Since pore size decreases moving down
this type of gel [4], polypeptides reach a “pore limit” at which they
can no longer migrate appreciably [5]. As such, unique migration
patterns can be achieved to optimize protein fractionation. The use
of gradient polyacrylamide concentrations in SDS-PAGE can offer
further advantages over fixed concentration gels, including 1) The
ability to separate a wider range of protein sizes in a single gel due
to the decreasing pore size as peptides migrate down the gel; 2)
larger effective range of acrylamide concentrations for gradient gels
(for fixed acrylamide concentration: 5e20%; for gradient: 3e30%)
[2]; 3) higher resolving power of distinct proteins, fragments, or
* Corresponding author.
E-mail address: enorstro@depaul.edu (E. Norstrom).
glycoforms with close molecular weights; 4) The leading edge of a
migrating protein is slowed more than the trailing edge, resulting in
sharper bands [6]. In addition, glycosylated proteins, which migrate
unpredictably in fixed-concentration SDS-PAGE gels, have been
observed to exhibit smaller migration variance in gradient gels [7].
Due to these advantages, separating proteins by gradient gel
SDS-PAGE has become a common practice in research and teaching
labs. However, the process of pouring gradient gels is a relatively
time-consuming process and has traditionally required equipment
such as a mixing apparatus, peristaltic pump, as well as tubing and
other materials, all of which must be cleaned after each pour to
prevent clogging and contamination of subsequent gels [2]. This is
especially important if gels requiring multiple buffer systems are
poured using the same apparatus (e.g. tris-glycine and tris-tricine).
Although many options are available from commercial suppliers,
these may not provide optimal ranges for a researcher's specific
needs, and the cost may be prohibitive for small labs or teaching
labs. Moreover, they may require proprietary buffer systems and
have limited shelf life. Lastly, certain downstream applications such
as silver staining are reported by some manufacturers to be less
effective in pre-cast gels than in hand poured gels [8].
Thus, there exists a need for a fast, affordable, and customizable
method to cast gels for gradient SDS-PAGE. The simplified protocol
presented here allows for the pouring of gradient and “stacked”
polyacrylamide gradient gels. It has been used effectively in our lab
to study the amyloid precursor protein (APP) and its many

http://dx.doi.org/10.1016/j.ab.2016.07.003
0003-2697/© 2016 Elsevier Inc. All rights reserved.
 A.J. Miller et al. / Analytical Biochemistry 509 (2016) 12e14 13

metabolites which range from a relative mobility of over 100kD for
the full length protein to less than 10kD [9]. Moreover, some of
these metabolites differ by only 2kD and require high resolving
power to visualize multiple metabolites on a single gel [10].
The method involves generating separate resolving gel buffers
differing in acrylamide concentration. The lower concentration gel
buffer is drawn into a serological pipet until a desired volume is
reached, e.g. half of the resolving gel volume. Using the same pipet,
the researcher draws up the higher acrylamide concentration until
the final gel volume is reached (Fig. 1A). When the gel solution is
ejected into the casting plate sandwich, the higher acrylamide
concentration is deposited on the bottom followed by the lower
concentration on top. This creates a “stacked” concentration
gradient with very little mixing of the phases at the interface. If
desired, some mixing of the interface can be achieved by allowing
one or two bubbles to flow up the pipet (Fig. 1B). The acrylamide
concentrations can vary as per the investigator's needs as can the
volume ratio of each concentration. This method can be used to
generate stacked gels of multiple acrylamide concentrations or a
modified linear gradient for routine analysis where a linear
gradient will provide additional benefits to the researcher but in
which a perfectly linear gradient is not necessary (For protocol and
description of migration see Ref. [11]). Other components of the
two phases can also be altered, for example the addition of glycerol
to the lower phase [12]. This protocol requires only standard gel
casting equipment along with a serological pipet and pipet
controller and can be used with any PAGE buffer system of the
investigator's choice. It can be used easily with minigel systems as
presented here, or investigators may adopt their own optimization
for their electrophoresis apparatus of choice. While the acrylamide
gradients resulting from this simplified method may not achieve
the near-perfect linearity of gels poured with a casting apparatus,
the gradient will be sufficient for many gradient gel PAGE appli-
cations and offers the advantages described above for routine
electrophoretic analysis. In many situations, the investigator may
wish to resolve multiple polypeptides with a wide range of relative
masses. In these cases, a “stacked” gradient gel can provide the
necessary fractionation ranges and resolution. For example, the
resolution of a large protein and its metabolites of smaller molec-
ular mass on the same gel can offer valuable information on the
relative levels of each metabolic product relative to the full-length
precursor. In this case, detection of components on the same gel
offers advantages when quantitation of relative levels is desirable.
2. Results
To compare methods of gradient gel production, broad range
molecular weight markers and HEK293 cell lysates were run on a
precast 4e15% tris-glycine gel (Bio-Rad), and a hand-poured 4e15%
simplified gradient gel for which the ratio was 40% of the volume
being 4% acrylamide and 60% of the volume being 15% acrylamide
(using a Bio-Rad Mini-PROTEAN system). The proteins were stained
with coomassie blue to visualize relative migration of the constit-
uents (Fig. 1C). The simplified gradient gel exhibits a broad
migration similar to that of the precast gel. In both cases, the
migration was distinct from that of a fixed 10% acrylamide gel run in
parallel for comparison. Note that the simplified gradient gel give
enhanced resolution in the lower and high molecular weight re-
gions relative to the fixed 10% gel. To demonstrate the even dis-
tribution of polypeptides across an entire gel, a whole gel image is
included in Fig. 2A. In practice we have not observed disruptions of
protein migrations using this method, i.e. each lane in the gel is
observed to have identical migration characteristics.
To demonstrate the utility of the simplified gradient in a
research setting, metabolites of the amyloid precursor protein
(APP) were analyzed using the simplified gradient method. APP
(UniProtKB: P05067) is involved in the pathogenesis of Alzheimer's
disease as the holoprotein from which the beta-amyloid (Ab)
peptide is derived [13]. However, Ab is only one of many relevant
cleavage products of APP. To study APP processing, several me-
tabolites of various sizes must be analyzed alongside the remaining
uncut full-length protein. Gels in our lab are often expected to

Fig. 1. Sketch of sample acrylamide concentration differentials within the pipet used to pour gel. Numbers represent mL marks on a standard 10 mL pipet. A) Note that buffer
containing lower acrylamide concentration (light gray) is aspirated into pipet first followed by higher concentration (dark gray). When liquid is ejected into gel sandwich, the high
concentration acrylamide is on the bottom. B) When a linear gradient is desired, a bubble is allowed to migrate up the pipet which will cause mixing at the phase interface. C)
Comparison of molecular weight standards and HEK293 cell lysate run on fixed 10% gel (left), a Bio-Rad pre-case 4e15% gradient gel, (center) and a simplified gradient (right).
14 A.J. Miller et al. / Analytical Biochemistry 509 (2016) 12e14
Fig. 2. A) Whole gel image of a 4e15% gel poured using the simplified gradient method. Gel loaded with Laemmli sample buffer (lanes 1, 4, 7, 10), HEK293 cell lysate [2,5,8] and
molecular weight protein standard [3,6,9]. B) Sample analysis of APP metabolites using a 10e16% tris-tricine gel. Gel can resolve full length APP as well as the C-terminal fragments
C99 and C83.
resolve multiple forms of full-length APP (APP is 78.7 kDa, but
exhibits complex glycosylation and migrates closer to 110 kDa on
SDS-PAGE) as well as multiple metabolites within cell lysates with
masses of 12, 10, and 4kD [14].
To achieve these separations, HEK cells stably expressing APP695,
the most abundant neuronal form of APP [15], were lysed and run
on 10e16% acrylamide gradient tris-tricine gels [16], made using
the “stacked” gel casting technique described above. Proteins were
transferred to PVDF membrane and probed with a C-terminal
antibody to detect fragments of APP containing the C-terminus
(Fig. 2B). The resultant gels resolve full-length APP as well as the
C83 and C99 metabolites e two different C-terminal fragments
resulting from differential processing by either a-secretase or b-
secretase cleavage, respectively. In this case, a “stacked” gradient
was used, which allowed for optimal separation of the full length
protein as well as the resolution of C83 and C99 with molecular
masses of 9.2kD and 11.1kD respectively.
Gels made using this simple protocol grant investigators more
options to customize their protein fractionation and allow optimal
resolution in multiple ranges on a single gel which can enhance
detection resolution in multiple methods requiring protein elec-
trophoresis, e.g. Western blot or proteomics. Thus, this technique
provides researchers and teachers without gradient mixing
equipment the agency to tailor their gels' concentrations, gradients,
and “stacked” gradients to their own specific research needs in less
time than traditional gradient mixing and without purchase of pre-
cast gels. Using this process, the pouring process requires less labor
and less time from lab workers, and the method is simple enough
for novice lab members to perform with little training. Moreover,
the risk of premature polymerization of high concentration acryl-
amide buffers before or during casting of the gel is removed with
this technique. When the relative volumes of the two buffers
containing different acrylamide concentrations is kept constant,
the technique has been wholly consistent with reproducible results
from assay to assay. In summary, the gradient gels made using this
simplified technique provide many of the advantages of true
gradient SDS-PAGE gels while significantly minimizing time, costs,
and complications of traditional in-house casting of gradient gels.
The technique is highly customizable to investigators' needs.
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