METHODOLOGICAL REPORT
ELECTROPHORETIC SEPARATION OF MYOSIN HEAVY
CHAIN ISOFORMS USING A MODIFIED MINI GEL
SYSTEM
MICHAEL D. ROBERTS,1 VINCENT J. DALBO,2 KYLE L. SUNDERLAND,3
1
Department of Biomedical Sciences, University of Missouri, Columbia, Missouri; 2Central Queensland University, Institute for
Health and Social Science Research, School of Medicine and Applied Sciences, Rockhampton, QLD, Australia; 3Department of
Health and Exercise Science, University of Oklahoma, Norman, Oklahoma; and 4Department of Health, Exercise, and Sports
Sciences, University of New Mexico, Albuquerque, New Mexico
ABSTRACT
Roberts, MD, Dalbo, VJ, Sunderland, KL, and Kerksick, CM.
Electrophoretic separation of myosin heavy chain isoforms
using a modified mini gel system. J Strength Cond Res 26(12):
3461–3468, 2012—The electrophoretic separation of myosin
heavy chain isoforms from muscle biopsy homogenates has
been widely practiced in the field of exercise physiology to
examine how intrinsic (i.e., aging) and extrinsic (i.e., training)
factors affect muscle phenotype. In the past, various research
groups have used large and mini polyacrylamide gel systems to
perform this delicate methodology. As technology has pro-
gressed, additional gel formats have been introduced, but avail-
able methodologies appear to be lacking. In this investigation,
we successfully separated 3 distinct myosin heavy chain iso-
forms from various muscle samples using a modified mini gel
system that can load up to 26 samples per gel. This article will
outline our allocated protocol and discuss potential trouble-
shooting considerations for other researchers performing this
intricate methodology. The outlined methodology has resulted
in an ability to clearly resolute 3 distinct bands at molecular
weights attributed to the myosin heavy chain isoforms in human
skeletal muscle at a wide range of human ages (20–78 years).
As additional technologies become available, the need to mod-
ify and adapt existing electrophoretic protocols for myosin
heavy chain isoform separation and other protocols will con-
tinue to be evident.
KEY WORDS myosin heavy chain isoforms, muscle biology,
exercise, gel separation, electrophoresis
Address correspondence to Chad Kerksick, chadkerksick@unm.edu.
26(12)/3461–3468
Journal of Strength and Conditioning Research
Ó 2012 National Strength and Conditioning Association
Copyright © National Strength and Conditioning Association Unauthorized reproduction of this article is prohibited.
AND CHAD M. KERKSICK4
INTRODUCTION
E
xercise physiologists have been very interested in
separating the 3 prominent myosin heavy chain
isoforms (i.e., type I, type IIa, and type IIx) using
gel electrophoresis given that this methodology is
used for muscle fiber typing (9) and determining how exer-
cise (1), aging (8), hormonal therapy (6), or other stimuli
affect this phenotype in humans and animals. Nonetheless,
this methodology presents itself with a unique set of chal-
lenges (i.e., electrophoresis equipment, run times, voltage
and current settings, stacking and separating gel composi-
tions including the acrylamide:bisacrylamide ratio, gradient
vs. nongradient separating gels, buffer ingredients, amount of
protein loaded, and staining procedure) making the process
a significant challenge, especially when the equipment evolves
and differs from laboratory to laboratory. Indeed, Hammed
et al. (5) were able to separate 3 prominent myosin heavy
chain isoform bands in only 1 of 15 human participants using
the mini Protean III (Bio-Rad Laboratories, Hercules, CA,
USA), and a well-established electrophoresis protocol in
a study that examined the effects of fiber type and resistance
exercise on mechanogrowth factor mRNA expression pat-
terns (4). Moreover, Kohn and Myburgh (7) achieved optimal
myosin isoform band separation with the addition of 2-mer-
captoethanol to the upper electrode buffer, whereas Blough
et al. (3) achieved virtually no band separation with severe
smearing without the addition of 2-mercaptoehtanol to the
upper electrode buffer. Finally, Bamman et al. (2) determined
that using a 7% separating gel vs. an 8% gel created myosin
bands that were too diffuse for analysis. Taking these few
reports into consideration, it seems obvious that minor devia-
tions from previously published protocols can lead to inappro-
priate band separation or virtually no band separation at all.
Historically, the studies available on separation of the
myosin heavy chain isoforms have used electrophoretic boxes
that at first were large (i.e., Bio-Rad Protean IIxi: 16 cm
[height] 3 16 cm [weight] 3 1 mm [thickness]) (1) and then
progressed to mini or smaller formats (i.e., Bio-Rad Mini Pro-
tean III, which is 8.0 cm [width] 3 7.3 cm [height] 3 0.75–1.0
VOLUME 26 | NUMBER 12 | DECEMBER 2012 | 3461
 Myosin Heavy Chain Isoforms and Electrophoresis
mm [thickness]) (7). An intermediate or modified mini gel
electrophoresis (Criterion Cell; catalog #165-6001, Bio-Rad
Laboratories) is available that can load up to 26 samples per
gel and 2 gels per run. Despite using previously allocated
protocols for mini gel systems (2,7), our research group expe-
rienced distinct challenges attempting to adequately perform
the electrophoretic separation of the 3 myosin heavy chain
isoforms from human muscle samples using this modified
system. Therefore, the purpose of this report is to outline
the resulting protocol we adapted that has led to an excep-
tional resolution of all 3 myosin heavy chain isoforms. In
addition, we seek to provide additional troubleshooting guide-
lines for future researchers who are looking to perform the
electrophoretic separation of myosin heavy chain isoforms
using electrophoresis equipment that may differ from what
is currently used by other laboratories. It is our hope that
researchers can practically apply our findings to perform these
experiments with similar electrophoresis equipment (i.e., high-
er throughput mini or intermediate sized electrophoresis
units) or use our findings to troubleshoot their experiments
with other electrophoresis equipment.
METHODS
Subjects and Tissue Treatment
The samples used in this study were collected as part of
another investigation whereby recreationally active male
participants between the ages of 20 and 80 years were
recruited to complete a series of fitness tests and a muscle
biopsy. It should be noted that the subjects in the afore-
mentioned study were informed of the experimental proce-
dures and signed informed consent statements and medical
history forms in adherence with the human subjects’ guide-
lines of the University of Oklahoma Health Sciences Center
Institutional Review Board and the American College of
Sports Medicine before any data collection.
Step 1: Preparing Muscle Samples
For this investigation, muscle samples weighing approxi-
mately 10 mg were obtained from the right vastus lateralis
of each participant and homogenized using 500 ml of cell lysis
buffer (Tris-HCl, pH 6.8, 5% 2-mercaptoethanol, 10% glycerol,
2.3% sodium dodecyl sulfate [SDS]) using tight-fitting plastic
pestles that fit into 1.5-ml microcentrifuge tubes. After muscle
homogenization, the samples were heated for 10 minutes at
608 C, and 200 ml of 100% glycerol was added to the samples
before storage at 2808 C for subsequent protein analysis. The
protein content of each sample was determined spectropho-
tometrically at a wavelength of 595 nm using Bradford reagent
(Bio-Rad Laboratories; Hercules, CA, USA) to standardize
the amount of protein loaded per well.
Step 2: Preparation of Buffers
Six separate runs were performed on 2 muscle homogenates
in duplicate as indicated in Table 1. Our methods were largely
derived from Kohn and Myburgh (7) with the exception of
buffer strength and voltage settings that will be described
the TM
3462 Journal of Strength and Conditioning Research
Copyright © National Strength and Conditioning Association Unauthorized reproduction of this article is prohibited.
below (step 3). The following describes the reagents that need
to be made and were used as part of these protocols to suc-
cessfully separate myosin heavy chain bands.
One hundred milliliters of 51.4% glycerol (to make gels):
51.4 ml of 100% glycerol (Sigma; catalog#: 65516, St. Louis,
MO, USA) was added to 48.6 ml of distilled H2O (dH2O) in
a media storage container. No pH adjustment was made.
One hundred milliliters of 30% acrylamide (49:1, acryl-
amide:bis; to make gels): 29.4 g of acrylamide powder
(Sigma; catalog#: A3553) was added with 0.6 g of bisacry-
lamide powder (Sigma; catalog#: M7279) in a media storage
container. Using dH2O, the total solution was made up to
100 ml; no pH adjustment was made.
One hundred milliliters of resolving gel Tris buffer (to make
gels): 16.2 g of Tris powder (Bio-Rad Laboratories; catalog#:
161-0716, Hercules) was added with 5.0 g of glycine powder
in a media storage container. Using dH2O, the total solution
was made up to 100 ml and 2.7 g of SDS powder then added.
Next, we used concentrated HCl (Sigma; catalog#: HX0603-4)
and a calibrated pH meter to adjust the pH of this solution to
8.80 6 0.05 units. Note that if the pH became too low, then we
used Tris powder to bring the pH back up to 8.80 6 0.05 units.
One hundred milliliters of stacking gel Tris buffer (to make
gels): 3.0 g of Tris powder (Bio-Rad Laboratories; catalog#:
161-0716) was added with 0.4 g of ethylenediamminetetraa-
cetate (EDTA, Sigma; catalog#: E9884) powder in a media
storage container. Using dH2O, the total solution was made
up to 100 ml, and subsequently, 1.4 g of SDS powder was
added to this solution. Next, concentrated HCl and a cali-
brated pH meter were used to adjust the pH of this solution
to 6.80 6 0.05 units. Again, if the pH became too low, Tris
powder was used to bring the pH back to 8.80 6 0.05 units.
Five milliliters of 10% ammonium persulfate (to make
gels): 5 ml of dH2O was added to 0.5 g of ammonium per-
sulfate powder (Sigma; catalog#: A3678). This solution
should be made fresh daily.
One liter of 103 electrophoresis buffer (500 mM Tris,
750 mM glycine, 0.5% SDS [pH: 9.0 6 0.05]): 60.6 g of Tris
powder (Bio-Rad Laboratories; catalog#: 161-0716) was
added with 56.3 g of glycine (Sigma; catalog#: 68898) pow-
der in a media storage container. The total volume was
brought to 1 L with dH2O and subsequently 5 g of SDS
powder was added to this solution.
Forty milliliters (per gel) of upper electrode buffer
(500 mM Tris, 750 mM glycine, 0.5% SDS [Sigma; Catalog#:
L4390], % 2-mercaptoethanol [no pH adjustment]): 48 ml of
2-mercaptoehtanol (Sigma; catalog#: M7154) was added to
40 ml of 103 myosin heavy chain (MHC) buffer in a 50-ml
graduated cylinder.
One liter of lower electrode buffer (100 mM Tris, 150 mM
glycine, 0.1% SDS [no pH adjustment]): 200 ml of 103
MHC buffer (see above) was added with 800 ml of dH2O.
One liter of destain solution: 75 ml of 1.0 M acetic acid
(Sigma; catalog#: 318590) was added to 400 ml of methanol
(Sigma; catalog#: M1775) and 525 ml of dH2O.

TABLE 1. Various electrophoresis conditions employed during runs.
Run Voltage and run times
1 6 h at 130 V at 48 C
(Amps: 0.06 / 0.02)
2 7.5 h at 135 V at 48 C
(Amps: 0.06 / 0.01)
3 10 h at 135 V at 48 C
(Amps: 0.06 / 0.01)
4 10 h at 200 V at 48 C
(Amps: 0.06 / 0.01)
5 10 h at 180 V at 48 C
(Amps: 0.06 / 0.01)
Copyright © National Strength and Conditioning Association Unauthorized reproduction of this article is prohibited.
the TM
Journal of Strength and Conditioning Research | www.nsca.com
Images (from left / right: myosin
from ladder, sample from a 20-y-old
male participant, sample from
Other conditions a 40-y-old man)
Stacking gel: 3% acrylamide (30:1
acrylamide:bisacylamide), 125 mM Tris,
pH 6.8, 1% SDS
Separating gel: 30% glycerol, 5%
acrylamide (30:1 acrylamide:
bisacylamide), 375 mM Tris, Two bands apparent per sample
pH 8.8, 1% SDS
Lower electrode buffer: 25 mM Tris, Bands were smeared and diffuse
192 mM glycine, 0.1% SDS
(pH not adjusted)
Upper electrode buffer: same as
lower electrode buffer
Amount of protein loaded per
well: 2 mg
All conditions same as in run 1
One band apparent per sample
Bands were smeared and diffuse
Stacking gel: 3% acrylamide (49:1
acrylamide:bisacylamide), 125 mM Tris,
pH 6.8, 1% SDS
Separating gel: 30% glycerol, 8%
acrylamide (49:1 acrylamide:
bisacrylamide), 375 mM Tris, Two bands apparent per sample
pH 8.8, 1% SDS
Lower electrode buffer: 25 mM Tris, Bands were smeared and diffuse
192 mM glycine, 0.1% SDS
(pH not adjusted)
Upper electrode buffer: same as
lower electrode buffer
Amount of protein loaded per well:
2 mg
Stacking gel: conditions same as
in run 3
Separating gel: conditions same 2–3 Bands apparent per sample
as in run 3
Lower electrode buffer: 50 mM Tris, Bands were smeared and diffuse
75 mM glycine, 0.05% SDS
(pH not adjusted)
Upper electrode buffer: 63 lower buffer
with 0.12% 2-mercaptoethanol added
Amount of protein loaded per well: 2 mg
Stacking gel: 30% glycerol, 4%
acrylamide (49:1 acrylamide:
bisacylamide), 70 mM Tris-HCl,
pH 6.8, 4 mM EDTA, 0.4%
SDS
(continued on next page)
VOLUME 26 | NUMBER 12 | DECEMBER 2012 | 3463
 Myosin Heavy Chain Isoforms and Electrophoresis

Separating gel: 30% glycerol, 8% 2–3 Bands apparent per sample

6 11 h at 190 V at 48 C
(Amps: 0.06 / 0.01)
acrylamide (49:1 acrylamide:bis-
acrylamide), 200 mM Tris-HCl, pH 8.8,
100 mM glycine, 0.4% SDS
Lower electrode buffer: 100 mM Tris, Use of 21 gauge syringe to remove
150 mM glycine, 0.1% SDS 10% overlay on right duplicate
(did not adjust pH) improved band resolution
Upper electrode buffer: 53 lower buffer
with 0.12% 2-mercaptoethanol added
Amount of protein loaded per well: 2 mg
Used 21 gauge needle on right duplicates
to remove 10% overlay of samples
before run
Stacking gel: conditions same as in run 5

Separating gel: conditions are the same 3 Bands apparent per sample
as in run 5
Lower electrode buffer: conditions are the
same as in run 5
Upper electrode buffer: conditions are the
same as in run 5
Amount of protein loaded per well: 1.5 mg
Used 21 gauge needle on both duplicates
to remove 10% overlay of samples
before run

Note that the voltage was fixed during each run, and the amperage varied slightly throughout the run but was not set.

Step 3: Hand-Casting Gels
Per the methods of Kohn and Myburgh (7), the separating
gel contained 30% glycerol, 8% acrylamide (49:1 acrylamide:
bisacrylamide), 200 mM Tris-HCl, pH 8.8, 100 mM glycine,
0.4% SDS. Similarly, the stacking gel contained 30% glycerol,
4% acrylamide (49:1 acrylamide:bisacrylamide), 70 mM Tris-
HCl, pH 6.8, 4 mM EDTA, 0.4% SDS as described by Kohn
and Myburgh (7). Blank 18-well Criterion Cell cassettes
(width: 14.5 cm, height: 8.3 cm, thickness: 1 mm; Bio-Rad
Laboratories; catalog#: 345-9902) were used to hand cast
gels. The separating gel was produced by mixing the follow-
ing: (a) 8.75 ml of 51.4% glycerol, (b) 4 ml of 30% acrylamide,
and (c) 2.25 ml of resolving gel Tris buffer in a beaker.
Specific instructions for producing these reagents have
been provided earlier in this article (step 2). Next, 150 ml of
10% ammonium persulfate (ingredients provided above) and
15 ml of tetramethylethylenediamine (TEMED) (Sigma;
catalog#: T9281) were added to the beaker, and the solution
was briefly swirled by hand; note that the solution was not
degassed under a vacuum as reported in other protocols.
Next, a 5-ml Eppendorf pipettor was used to deposit the
gel into the blank 18-well Criterion Cell one-quarter inch
below the wells and the gels. Approximately 2 ml of dH2O
was slowly applied using a 5-ml Eppendorf pipettor to over-
the TM
lay the gel in an effort to facilitate level polymerization of the
gel. Polymerization of the gel should begin within 2 minutes
and get completed after 1 hour.
After polymerization of the separating gel, the water
overlay was discarded, and the stacking gel was added.
The stacking gel was produced by mixing (a) 2.19 ml of
51.4% glycerol, (b) 0.5 ml of 30% acrylamide, and (c) 1.06 ml
of stacking gel Tris buffer in a beaker.
Specific instructions for producing these reagents have
been provided earlier in this article. Next, 75 ml of 10%
ammonium persulfate (ingredients above) and 7.5 ml of
TEMED (Sigma; catalog#: T9281) were added to the bea-
ker, and the solution was briefly swirled by hand; note, that
the solution was not degassed under a vacuum as reported in
other protocols. A 5-ml Eppendorf pipettor was again used
to deposit the gel into the blank 18-well Criterion Cell cas-
sette above the polymerized separating gel, the 18-well
divider comb was inserted, and the stacking gel was left to
polymerize for 1 hour.
Step 4: Performing Runs
After polymerization of the stacking gel, the hand-cast gels
were placed in the electrophoresis box. One liter of Lower
Tris buffer (from step 2) was placed in the electrophoresis box
and 40 ml of Upper Tris buffer (ingredients above) was placed

3464 Journal of Strength and Conditioning Research

Copyright © National Strength and Conditioning Association Unauthorized reproduction of this article is prohibited.

Figure 1. Voltage considerations for sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) gels when considering
gel dimensions.
in the upper chamber of each gel. After buffer placement, the
comb inserts of each hand-cast gel were removed.
Homogenized muscle samples were obtained from the freezer
and allowed to thaw at room temperature. One empty 1.2-ml
microcentrifuge tube per sample was used to place enough
sample and Laemmli reducing buffer (Bio-Rad Laboratories;
catalog#: 161-0737) spiked with 5% 2-mercaptoethanol (i.e., 950
ml of Laemmli buffer + 50 ml of 2-mercaptoethanol) into each
tube whereby 1.5 mg of protein per 30 ml of reducing buffer was
loaded per lane. These samples were then heated for 2 minutes
at 1008 C, and 30 ml of each sample was subsequently loaded
into each well using gel loading tips. Similarly, a broad-range
prestained molecular weight ladder (Bio-Rad Laboratories;
catalog#: 161-0318) was loaded in an adjacent lane to monitor
the migration rate of myosin, and the outside lanes were not
used. To improve band resolution, a 21-gauge hypodermic
needle was used to remove a small amount of overlay
(i.e., 10–20%) from each sample. Finally, the electrophoresis
box was plugged into the power source, the box was placed
in a refrigerator at 48 C, and successful runs were performed at
190 V and constant amperage (i.e., the runs started at 0.07 A and
ended at 0.01 A) for 11 hours.
Figure 2. Comparison of runs with gels that were premade and incubated for 1–3 days at 48 C before the run (left inset) vs. premade fresh before the run
(right inset).
Copyright © National Strength and Conditioning Association Unauthorized reproduction of this article is prohibited.
the TM
Journal of Strength and Conditioning Research | www.nsca.com
Step 5: Staining Gels and Performing Band Densitometry
After all the runs, the cassettes were opened, and the gels
were placed in a container filled with dH2O. The gels were
allowed to stand in dH2O for 1.5 hours so that SDS was
effectively removed from the gel and background staining
was reduced. Next, the dH2O was decanted from the hold-
ing container and Coomassie R250 (Bio-Rad; catalog#: 161-
D436) was added, and the gels were stained for 30 minutes.
Finally, the Coomassie Blue reagent was decanted, and the
gels were destained with destain solution (ingredients above)
in the following manner: (a) quick destain for 5 minutes, (b)
second destain for 30 minutes, (c) a third destain for 3.5
hours, and (d) and optional last destain for 1+ hours. The
gels were then placed in a gel documentation system
(Chemi-Doc XRS, Bio-Rad Laboratories; catalog#: 170-
8265) whereby the gel was imaged and bands were automat-
ically detected and analyzed using a computer software
package (Quantity One, Bio-Rad Laboratories; catalog#:
170-9600).
RESULTS
As mentioned, we separated and identified 3 distinct myosin
isoforms during runs set at 190 V, which lasted 11 hours.
Further, the separating gel employed during successful runs
contained 30% glycerol, 8% acrylamide (49:1 acrylamide:
bisacrylamide), 200 mM Tris-HCl, pH 8.8, 100 mM glycine,
0.4% SDS, whereas the stacking gel contained 30% glycerol,
4% acrylamide (49:1 acrylamide:bisacrylamide), 70 mM Tris-
HCl, pH 6.8, 4 mM EDTA, 0.4% SDS as described by Kohn
and Myburgh (7). Finally, the upper chamber buffer con-
tained 500 mM Tris, 750 mM glycine, 0.5% SDS, 0.12%
2-mercaptoethanol (did not adjust pH), whereas the lower
electrode buffer contained 100 mM Tris, 150 mM glycine,
0.1% SDS (did not adjust the pH). Table 1 outlines other
runs in which different experimental conditions were
employed.
During runs 1 and 2, the stacking gel contained 3%
acrylamide (30:1 acrylamide:bisacrylamide), 125 mM Tris,
VOLUME 26 | NUMBER 12 | DECEMBER 2012 | 3465
 Myosin Heavy Chain Isoforms and Electrophoresis
pH 6.8, 1% SDS, whereas the separating gel contained 30%
glycerol, 5% acrylamide (30:1 acrylamide:bisacrylamide),
375 mM Tris, pH 8.8, 1% SDS. Further, the lower and upper
electrode buffers contained 25 mM Tris, 192 mM glycine,
0.1% SDS (pH not adjusted). Finally, run times varied from
6 to 7.5 hours at 130–135 V. We found these conditions to
yield incomplete separation of myosin bands and extensive
smearing.
During runs 3 and 4, the stacking gel contained 3%
acrylamide (49:1 acrylamide:bisacrylamide), 125 mM Tris,
pH 6.8, 1% SDS, whereas the separating gel contained 30%
glycerol, 8% acrylamide (49:1 acrylamide:bisacrylamide),
375 mM Tris, pH 8.8, 1% SDS. During run 3, the lower
and upper electrode buffers contained 25 mM Tris, 192 mM
glycine, 0.1% SDS (pH not adjusted). During run 4,
the electrode buffers were more highly concentrated, and
2-mercaptoethanol was added to the upper electrode buffer
(i.e., lower electrode buffer: 50 mM Tris, 75 mM glycine,
0.05% SDS [pH not adjusted]; upper electrode buffer: 63
lower buffer with 0.12% 2-mercaptoethanol added). When
we applied 135 V for 10 hours, concomitant with the afore-
mentioned conditions during run 3, 2 faint isoform bands
appeared, which were poorly resolved. When applying
200 V for 10 hours during run 4, a third myosin band was
beginning to appear, albeit the bands had smeared.
During runs 5 and 6, we increased the upper and lower
chamber buffer strengths (mentioned above) based on the
notion that this alteration may increase band resolution. We
developed a linear regression equation that predicted the
voltage that needed to be applied based the voltages used for
gels that varied in size in other literature (7) as depicted in
Figure 1.
Finally, we found that performing these runs from 10 to 11
hours was enough to allow the prestained myosin molecular
weight marker to migrate 60–70% down the stacking gel,
which has also been reported in the literature (2).
DISCUSSION
The purpose of this methodological study was to outline
the resulting protocol we adapted that has led to excep-
tional resolution of all 3 myosin heavy chain isoforms using
a modified mini electrophoresis system. In addition, we
wanted to provide additional troubleshooting guidelines for
future researchers who are looking to perform the electro-
phoretic separation of myosin heavy chain isoforms. Three
distinct myosin isoforms were identified using the following
conditions: (a) 190 V for 11 hours (amperage started at
0.06 A and dropped to 0.01 A), (b) the separating gel
contained 30% glycerol, 8% acrylamide (49:1 acrylamide:
bisacrylamide), 200 mM Tris-HCl, pH 8.8, 100 mM glycine,
0.4% SDS, whereas the stacking gel contained 30% glycerol,
4% acrylamide (49:1 acrylamide:bisacrylamide), 70 mM
Tris-HCl, pH 6.8, 4 mM EDTA, 0.4% SDS as described
by Kohn et al. (7), and (c) the upper chamber buffer con-
tained 500 mM Tris, 750 mM glycine, 0.5% SDS, 0.12%
the TM
3466 Journal of Strength and Conditioning Research
Copyright © National Strength and Conditioning Association Unauthorized reproduction of this article is prohibited.
2-mercaptoethanol, whereas the lower electrode buffer
contained 100 mM Tris, 150 mM glycine, 0.1% SDS. No
pH adjustment was made for either the upper or lower
buffers. We found that using these conditions yielded an
adequate 3-band separation of myosin heavy chain iso-
forms from the muscle homogenates of human skeletal
muscle.
We demonstrated that 5% acrylamide (30:1 acrylamide:
bisacrylamide) was unsuitable for band separation and
resolution using our electrophoresis system. As mentioned,
Bamman et al. (2) determined that using a 7% separating
gel vs. an 8% gel created myosin bands that were too
diffuse for analysis. These authors stated that they had
attempted to use 7% gels to increase the electrophoretic
mobility of proteins through the gel, albeit employing this
slight alteration to the acrylamide percentage caused
smearing. Hence, the findings of this report are in agree-
ment with our findings in that (a) 8% acrylamide is suitable
for separating gels and (b) decreasing this percentage
causes band smearing, which inhibits the identification of
3 myosin isoforms.
Of further interest is the usage of acrylamide, which
contains a 49:1 acrylamide:bisacrylamide ratio. We had
initially allocated acrylamide that contained a 30:1 acrylam-
ide:bisacrylamide ratio during runs 1–4. However, we decided
to alter this composition to the former based on the conten-
tion of Kohn and Myburgh (7) who stated the following:
Myosin heavy chain proteins are very large (;220
kDa), and conventional SDS-PAGE fails to separate
the isoforms into distinct bands, mostly as a result of
the low acrylamide:bisacrylamide ratio (37.5:1).
Hence, this is another methodological alteration that
resulted in the successful isolation of 3 distinct isoform
bands and an aspect that should be considered by labora-
tories that may struggle with this methodology.
One major alteration employed that differed from pre-
vious investigations using mini gel systems was altering the
voltage from 140 to 190 V (constant voltage, varying
amperage) during the 10- to 11-hour runs. During our
earlier runs, lower voltages (120 V; data not shown) were
employed with minimal success indicated by low currents
(i.e., 0.01 A to run stoppages because of a lack of current)
and minimal band migration down the separating gel.
Regardless, the fact that Kohn and Myburgh (7) employed
different voltages for differently sized gels prompted our
group to calculate the area of the gel and adjust the voltage
relative to the previous voltages used for other gel systems
and sizes (per Figure 1). As indicated in Table 1, this modest
alteration allowed for the prestained myosin molecular
weight marker to migrate approximately 70% down the
stacking gel within a 10- to 11-hour period and yielded
proper band separation in the muscle specimens after Coo-
massie staining. In this regard, future researchers employing

this technique should ensure that the voltage setting during
electrophoresis runs yield an adequate current.
Another major alteration employed by our group that
differed from Kohn et al. was the buffer concentrations
employed during the respective experiments. In general,
the conductivity of electrophoresis buffers is proportional
to the ionic strength, or the capacity for the solution to
ionize and conduct and electrical current. Interestingly, we
discovered that applying the same upper electrode buffer
as Kohn and Myburgh (7) yielded poor electrophoretic
results given that this is a relatively low conductive buffer.
In contrast, increasing the strength (i.e., concentrations of
Tris, glycine, and SDS) of the upper electrode buffer by
67% (i.e., from 6-fold of the lower electrode buffer used
by Kohn et al. to 10-fold of the lower electrode buffer used
by this group) and the lower electrode buffer by 50%
yielded appropriate conductivity conditions. In short, we
found this seemingly minute methodological difference to
be integral to proper isoform separation. In this regard,
investigators performing this technique in future studies
may perform pilot experiments to find the optimal buffer
concentrations that fit their electrophoresis equipment.
An integral methodological consideration when per-
forming the electrophoretic separation of myosin heavy
chain isoforms is making the gels fresh immediately
before using them. A major inexplicable problem that
alluded our laboratory was smearing that had occurred
during runs in which gels were stored 1–3 days at 48 C
(Figure 2). In this regard, we repeatedly found that using
gels that were stored after polymerization yielded poor
separation compared with gels that were made before
the run.
Finally, we loaded 1.5–2.0 mg of protein into each lane during
successful electrophoresis runs. Up to 4.0 mg of protein (0.13
mg/ml) was loaded during prior experiments, albeit this sample
concentration overloaded the Criterion wells and produced
a protein bands that were too dense for the determination of
3 bands (data not shown). Taking our data into consideration
suggests that as little as 0.05 mg/ml (1 mg of protein per sample)
of myofibrillar protein can be used to determine fiber typing
using myosin heavy chain electrophoresis.
CONCLUSIONS
In summary, we propose that this aforementioned tech-
nique successfully isolated 3 distinct myosin heavy chain
bands from human skeletal muscle homogenates. Note that
this method has been successfully performed by Staron’s
group (4) and other researchers. Therefore, the electropho-
retic separation of myosin bands itself in this article is not
novel. Nonetheless, the adjustments made because of our
modified electrophoresis system do create the need for
important considerations for successful run performance.
Researchers who are experiencing problems with this tech-
nique should think about the following major considera-
tions: (a) gels are made fresh before electrophoresis; (b) if
Copyright © National Strength and Conditioning Association Unauthorized reproduction of this article is prohibited.
the TM
Journal of Strength and Conditioning Research | www.nsca.com
gel systems differ from the mini Protean III or Criterion cell
(Bio-Rad International), then buffer strengths and voltages
should be selected according to the dimensions of the sys-
tem whereby it is recommended that the direction and
magnitude of changes in this study be first considered to
eliminate as much wasted time and sample as possible; (c)
8% acrylamide gels are used with acrylamide:bisacrylamide
ratio of 49:1 because this has been successfully allocated by
our group and others (2,7); and (d) the amount of protein
loaded (i.e., 0.05–0.1 mg/ml) is appropriate for optimal band
separation and greater amounts (0.4 mg/ml) are likely to
overload each lane and cause smearing.
PRACTICAL APPLICATIONS
Human skeletal muscle is composed of 3 distinct myosin
heavy chain isoforms that respond to internal stimuli such
as aging and external stimuli such as physical activity,
specifically heavy resistance training. Commonly associ-
ated phenotypes are linked with various myosin heavy
chain isoforms and the ability to identify the extent to
which these bands are expressed has been and continues to
be an important methodological approach for muscle
biologist and exercise scientists. The outlined methodology
has resulted in an ability to clearly resolute 3 distinct bands
at molecular weights attributed to the myosin heavy chain
isoforms in human skeletal muscle at a wide range of
human ages (20–78 years). This methodological article is
meant to serve as a reference to muscle biologists due to the
fact that this protocol has been challenging to researchers
in exercise science. Furthermore, we have determined that
the need to modify and adapt existing electrophoretic pro-
tocols for myosin heavy chain isoform separation is evident
given the advancement in the dimensions and throughput
of electrophoretic systems.
ACKNOWLEDGMENTS
The authors would like to thank the subjects who
participated in the study in which the muscle samples
were used. They would also like to gratefully thank the
reviewers for taking time to critique this manuscript. This
study was funded by a Young Investigator grant from the
National Strength and Conditioning Association and a Uni-
versity of Oklahoma Big XII fellowship awarded to Chad
Kerksick, PhD.
REFERENCES
1. Adams, GR, Hather, BM, Baldwin, KM, and Dudley, GA. Skeletal
muscle myosin heavy chain composition and resistance training.
J Appl Physiol 74: 911–915, 1993.
2. Bamman, MM, Clarke, MS, Talmadge, RJ, and Feeback, DL.
Enhanced protein electrophoresis technique for separating human
skeletal muscle myosin heavy chain isoforms. Electrophoresis 20:
466–468, 1999.
3. Blough, ER, Rennie, ER, Zhang, F, and Reiser, PJ. Enhanced
electrophoretic separation and resolution of myosin heavy chains in
mammalian and avian skeletal muscles. Anal Biochem 233: 31–35, 1996.
VOLUME 26 | NUMBER 12 | DECEMBER 2012 | 3467
 Myosin Heavy Chain Isoforms and Electrophoresis
4. Fry, AC, Allemeier, CA, and Staron, RS. Correlation between
percentage fiber type area and myosin heavy chain content in human
skeletal muscle. Eur J Appl Physiol Occup Physiol 68: 246–251, 1994.
5. Hameed, M, Orrell, RW, Cobbold, M, Goldspink, G, and
Harridge, SD. Expression of IGF-I splice variants in young and old
human skeletal muscle after high resistance exercise. J Physiol 547:
247–254, 2003.
6. Harjola, V, Jankala, H, and Harkonen, M. Myosin heavy chain
mRNA and protein distribution in immobilized rat skeletal muscle
are not affected by testosterone status. Acta Physiol Scand 169:
277–282, 2000.
the TM
3468 Journal of Strength and Conditioning Research
Copyright © National Strength and Conditioning Association Unauthorized reproduction of this article is prohibited.
7. Kohn, TA and Myburgh, KH. Electrophoretic separation of human
skeletal muscle myosin heavy chain isoforms: The importance of
reducing agents. J Physiol Sci 56: 355–360, 2006.
8. Short, KR, Vittone, JL, Bigelow, ML, Proctor, DN, Coenen-
Schimke, JM, Rys, P, and Nair, KS. Changes in myosin heavy
chain mRNA and protein expression in human skeletal muscle
with age and endurance exercise training. J Appl Physiol 99:
95–102, 2005.
9. Talmadge, RJ and Roy, RR. Electrophoretic separation of rat skeletal
muscle myosin heavy-chain isoforms. J Appl Physiol 75: 2337–2340,
1993.