Laboratory Exercise
The Experimental Teaching Reform in
Biochemistry and Molecular Biology for
Undergraduate Students in Peking University
Health Science Center
From the Department of Biochemistry and Molecular Biology, Peking
University Health Science Center, Beijing 100191, Peoples Republic of
China
Abstract
Since 2010, second-year undergraduate students of an eight-
year training program leading to a Doctor of Medicine degree
or Doctor of Philosophy degree in Peking University Health
Science Center (PKUHSC) have been required to enter the
Innovative talent training project. During that time, the stu-
dents joined a research lab and participated in some original
research work. There is a critical educational need to prepare
these students for the increasing accessibility of research
experience. The redesigned experimental curriculum of bio-
chemistry and molecular biology was developed to fulfill
such a requirement, which keeps two original biochemistry
experiments (Gel filtration and Enzyme kinetics) and adds a
new two-experiment component called Analysis of anti-
tumor drug induced apoptosis. The additional component,
also known as the project-oriented experiment or the
Keywords: experimental teaching reform; project-oriented
experiment; Western blotting; DNA ladder assay
Background
Undergraduates engaged in research experiences have been
demonstrated to show an increase in understanding, confi-
dence, and awareness and in their ability to think and
work like a scientist [14]. Since 2010, undergraduate stu-
dents of an eight-year training program leading to a Doctor
of Medicine degree (M.D.) or Doctor of Philosophy degree
Volume 43, Number 6, November/December 2015, Pages 428433
*Correspondence to: Department of Biochemistry and Molecular
Biology, Peking University Health Science Center, 38 Xue Yuan Road,
Beijing 100191, China. E-mail: juhuani@bjmu.edu.cn.
Received 10 March 2015; Revised 2 June 2015; Accepted 29 June
2015
DOI 10.1002/bmb.20890
Published online 7 October 2015 in Wiley Online Library
(wileyonlinelibrary.com)
428
Xiaohan Yang
Luyang Sun
Ying Zhao
Xia Yi
Bin Zhu
Pu Wang
Hong Lin
Juhua Ni*
comprehensive experiment, consists of Western blotting
and a DNA laddering assay to assess the effects of eto-
poside (VP16) on the apoptosis signaling pathways. This
reformed laboratory teaching system aims to enhance
the participating students overall understanding of impor-
tant biological research techniques and the instrumenta-
tion involved, and to foster a better understanding of the
research process all within a classroom setting. Student
feedback indicated that the updated curriculum helped
them improve their operational and self-learning capabil-
ity, and helped to increase their understanding of theoret-
ical knowledge and actual research processes, which laid
the groundwork for their future research work. VC 2015 by
The International Union of Biochemistry and Molecular
Biology, 43:428433, 2015.
(Ph.D.) in Peking University Health Science Center (PKUHSC)
have been required to enter research labs to conduct origi-
nal research in their second year, which is called
Innovative talent training project. Consequently, there is a
critical educational need to prepare these undergraduates
for the increasing accessibility of research experiences. The
traditional laboratory course of biochemistry and molecular
biology taught at PKUHSC prior to 2010 was comprised of
independent experiments, each designed to teach common
biochemical techniques rather than expose students to a
better understanding of the research processes. The call for
our curriculum change increased intensely. Therefore, we
set out to create a new curriculum which served as required
preparation for the second year program of these under-
graduate students as well as laid the groundwork for their
further studies in biochemistry and molecular biology [57].
Our traditional experimental curriculum is composed
of two biochemistry experiments (Gel-filtration, Enzyme
Biochemistry and Molecular Biology Education

Schedule of experiments in Department of
TABLE I Biochemistry and Molecular Biology
After the
Before the reform reform
Independent 6 experiments 2 experiments
experiments
Gel-filtration Gel-filtration
Enzyme kinetics Enzyme kinetics
SDS-PAGE
Small scale plasmid
extraction
Restriction enzyme
digestion of plasmid
PCR
Comprehensive 0 experiments 2 experiments
experiments
Western blotting
DNA ladder assay
kinetics) and four molecular biology experiments [sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE), small scale plasmid extraction, restriction enzyme
digestion of plasmid, and PCR]. It was designed to help stu-
dents test the basic theory; deepen their perceptual knowl-
edge; become familiar with common equipment; and tech-
niques and master basic experimental skills. Here, we
describe a modification of our traditional curriculum which
keeps the two traditional biochemistry experiments (Gel fil-
tration and Enzyme kinetics), discards the four independent
molecular biology experiments (SDS-PAGE, small scale
plasmid extraction, restriction enzyme digestion of plasmid,
and PCR) and adds a project-oriented component called
Analysis of anti-tumor drug induced apoptosis (Table I).
Apoptosis, also called programmed cell death, is a fun-
damental and complex biological process that enables an
organism to kill and remove unwanted cells during animal
development, normal homeostasis, and disease [8]. Analysis
of apoptosis is closely related to cancer biology and is a
very large area of current research, so we expected this
topic to arouse the learning interest of undergraduate stu-
dents and facilitate their preparation for the real research
process. The additional component in our curriculum,
also known as the project-oriented experiment or
comprehensive experiment, consists of Western blotting
and a DNA laddering assay to assess the effects of etopo-
side (VP16) on the apoptotic signaling pathways. This
project-oriented course focuses on solving a scientific prob-
Yang et al.
lem using current molecular biology techniques which
allows students to gain practical, hypothesis-driven lab
experience [911].
Western blotting is a widely used laboratory technique
that uses a specific antibody as a probe to detect and con-
firm the presence, absence, or abundance change of the cor-
respondent antigen in a mixed protein sample. The Western
blotting technique owes its sensitivity and specificity to two
big contributing factors, one is the high resolution of SDS-
PAGE and the other is the strong specificity of the solid
phase immune reaction, which permits its ability to detect
as little as 0.1 nanograms of target protein in a sample. Cas-
pases, a group of intracellular proteases, are responsible for
the deliberate disassembly of the cell into apoptotic bodies
during apoptosis and Caspase-3 is a critical executioner of
apoptosis [1214]. Activation of Caspase-3 requires proteo-
lytic processing of its inactive zymogen into activated p17
and p12 fragments, which have been generally acknowl-
edged as one of the apoptosis markers. Here we describe
the use of Western blotting to detect increased level of
cleaved Caspase-3 (p17) after treatment of HL-60 (Human
promyelocytic leukemia cells) cell line with etoposide
(VP16), a kind of anti-tumor drugs. In addition, apoptosis is
characterized by the activation of endogenous endonucle-
ases with subsequent cleavage of chromatin DNA into inter-
nucleosomal fragments of roughly 180 base pairs (bp) and
multiples thereof (360, 540, etc.). The DNA laddering assay
can be used to visualize this feature by separating cleaved
DNA fragments through gel electrophoresis [15].
Our redesigned 4-week laboratory course meets once a
week with class sizes of 1824 students per section. Each
school year, eleven or twelve sections are taught which
involves 240 undergraduate students from the eight-year
M.D./Ph.D. program. Gel filtration or Enzyme kinetics can
be finished within 4 class hours (half day), whereas West-
ern blotting analysis and the DNA ladder assay require 12
class hours (one and a half days) and 8 class hours (one
day) separately. Students work in teams of 2 or 3 and
members of the team must divide the tasks and develop a
strategy for completing the analyses. Student evaluation is
based on both a journal-style written lab report and their
performance in the experimental class.
Procedure
Western Blotting Analysis of Activation of
Caspase-3
HL-60 cells were cultured 2 weeks before the class by our
instructors. For each group of students, HL-60 cells were
split into two 3.5-cm dishes. When the cell density reached
60%, Dimethyl sulfoxide (DMSO) or VP16 (20 lM) was
added to the culture medium of cells. Forty-eight hours (h)
later, cells were washed by ice cold phosphate buffered
saline (PBS) and collected. The pelleted cells were stored at
2808C until use.
429
 Biochemistry and
Molecular Biology Education

antibody (A5316, Sigma, St. Louis, Missouri, United States)

Time schedule of Western blotting overnight at 48C followed by incubation with a secondary
TABLE II
antibody for 1 h at room temperature. Immunoreactive
bands were visualized using Western blotting Luminol rea-
Western gent (Santa Cruz Biotechnology, Dallas, Texas, United
blotting Time (h) Procedures States) on a Versadoc. The detailed timeline of Western
blotting is shown in Table II.
The first day 2 Cell lysis, Determination
(8:0016:00) of protein concentration, DNA Ladder Assay for Apoptosis
Preparing samples, HL-60 cells (10-cm dishes) were treated with DMSO or
Pouring the gels VP16 (20 lM) for 48 h by our instructors just before the
2 Loading the samples class. The DNA ladder assay was conducted as follows. On
and running the the day of the class, cells were washed in PBS and collected
gels/Lunch by centrifugation (1,500 3 g, 5 min). About 100 lL of lysis
buffer (1% NP-40 in 20 mM EDTA, 50 mM Tris-HCl pH 7.5)
0.5 Preparing the gel
was added and the suspension was vortexed vigorously for
sandwich
10 s (lysis of cells) and centrifuged (5,000 rpm, 5 min) to
1 Blotting transfer obtain the supernatant. About 10 lL of 10% SDS solution
1 Blocking was added to the collected supernatant (final: 1% SDS) and

overnight Primary antibody

incubation
The second day 0.5 Washing
(8:0011:30)
1 Secondary antibody
incubation
0.5 Washing
0.5 Detection

On the first day of the class, cells were lysed in 200 lL

lysis buffer [50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-
40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate
(SDS), 1 mM EDTA, 1 mM phenylmethanesulfonylfluoride
(PMSF), 13 protease inhibitor cocktails (add just before
use)] on ice for 30 min. Whole cellular proteins were
extracted by collecting the supernatants after centrifuging
cell lysates at the maximum speed for 10 min. Protein con-
centration was determined using the bicinchoninic acid
assay (BCA assay).
The appropriate volume of 63 SDS-PAGE loading
buffer was added to each sample and boiled for 10 min.
Cell lysates with equal concentration of protein were
resolved using 15% SDS-PAGE gels and transferred onto
nitrocellulose membranes. Blocking of non-specific binding
was achieved by placing the membrane in 5% non-fat milk
in 1% Tween-20 of Tris-buffered saline (TBS-T). Then,
membranes were incubated with either anti-Caspase 3
antibody (#9662, Cell Signaling, Danvers, Massachusetts,
United states) which detects endogenous levels of full
length Caspase-3 (35 kDa) as well as the large fragment of
Caspase-3 resulting from cleavage (17 kDa), or anti-b-actin
Western blot analysis of extracts from HL-60
FIG 1 cells, untreated or etoposide-treated (20 lM, 48
h), using Caspase-3 antibody. Beta-actin was
used as an internal control. (a) Three groups of
students loaded their samples on the same gel.
After the electrophoresis, separated proteins
were transferred to the nitrocellulose membrane.
Before the incubation of primary antibody, the
membrane was cut into two parts according to
the molecular weight and incubated with
Caspase-3 and b-actin antibodies separately. The
protein standards are indicated on the right side
of the image. (b) Western blot analysis of the
activation of Caspase-3 in a preliminary experi-
ment conducted by instructors.

430 Experimental Teaching Reform in Eight-Year M.D./Ph.D. Programs

 sulfate 90 lL, TEMED 10 lL), 23 lL of each sample is
resolved by electrophoresis for 1 or 2 h under 100 V. The
gel was gently removed from the glass plate and stained
with silver staining buffer (0.2% AgNO3, 1% glacial acetic
acid, 10% ethanol) in a clean plate for 15 min. Developer
working solution (3% NaOH, 0.5 mL formaldehyde/100 mL)
was added and incubated for 5 min until DNA bands
appeared. Developer solution was replaced with prepared
stop solution (5% acetic acid), and the gel was washed
briefly before capturing the image using Gel Logic camera.

DNA laddering assay of apoptosis. Three groups
FIG 2
of students loaded their samples on the same
TBE-PAGE. After the electrophoresis, the gel was
silver stained. The image was captured using Gel
Logic camera.
Hazards
PKUHSC Laboratory Chemical Safety Manual must be read
by every student before they begin the lab course. Students
are specifically warned about the toxicity of compounds
and reagents including etoposide, PMSF, protease inhibitor
cocktails, acrylamide storage solution, and silver staining
buffer which will be used in the class. In our case, we have
made the wearing of disposable gloves mandatory in the
handling of those solutions and provide designated contain-
ers for the disposal of contaminated waste.

then treated with 10 lL of 50 mg/mL RNase A (final 5 lg/

lL) for 2 h at 568C. About 10 lL of 25 mg/mL Proteinase K
(final 2.5 lg/lL) was added and incubated for 2 h at 378C
followed by addition of 1/2 volume (65 lL) of 10 M ammo-
nium acetate and 2.5 volumes (500 lL) of ice-cold ethanol.
After thoroughly mixing, the samples were incubated for 1
h at 2208C (ethanol precipitation). Samples were centri-
fuged for 20 min at 12,000 rpm, washed with 200 lL of
80% ice-cold ethanol and air-dried for 10 min at room tem-
perature. The pellet was dissolved in 20 lL of TE buffer.
After assembly of the vertical plate electrophoresis appara-
tus and preparation of the TBE-PAGE (H2O 5.2 mL, 53
TBE 2 mL, 30% PAGE 2.7 mL, 10% ammonium peroxydi-
Implementation
One week before the class, the instructors encouraged the
students to learn how the Caspase pathway of apoptosis
works as well as all known methods used to analyze apo-
ptosis by reading primary literature. They were asked to
discuss the caspase pathway during the incubation times of
the experiments (for electrophoresis, transfer, blocking,
etc) instead of the instructors introducing these concepts to
them [16]. Motivated by self-learning, the students were
very active in the class. The instructors also took advantage
of the experimental incubation times to show students the
cell culture room with Powerpoint and introduced them

Student survey results

TABLE III

How would you describe the

gain you made in: No gain Little gain Moderate gain Good gain Great gain

General technical lab skills 0 0 6 49 1

Understanding the mechanism of Western 0 0 0 0 56
blotting and DNA ladder assay
Understanding the apoptosis signal pathway 0 0 5 38 13
Ability to cooperate 0 0 26 25 5
Conceptual problem-solving skills 0 0 23 29 4
Ability to interpret experimental results 0 2 10 36 8
Helping you to prepare better for the 0 0 16 32 8
innovation personnel training plan

Yang et al. 431


basic cell culturing techniques with pictures on slides. This
was due to the inaccessibility of all students to conduct the
cell culturing steps of the experiment. We also introduced
in detail the mechanism of how SDS-PAGE separates pro-
teins of different molecular weight and discussed each step
of Western blotting. This section was dedicated to under-
standing the role of SDS-PAGE working components (buffer
and pH), the information on how to run and program the
equipment, and the basic troubleshooting ideas. The mech-
anism of the DNA ladder assay is relatively simple to be
understood, so we placed emphasis on offering key tips on
the experimental procedure. The students were reminded
that although the course was designed with maximal suc-
cessful outcome ratios, in an actual research laboratory
setting, they must deal with experiments that take
extended timeframes to complete multiple trials for optimi-
zation, and work in collaboration with other people to opti-
mize equipment and reagent use.
Students recorded their results and analyzed their data
in detail in their reports. As previously mentioned, VP16
treatment leads HL-60 cells to apoptosis, which results in
the increased cleaved Caspase-3. As expected, the students
observed significantly increased cleaved Caspase-3 (p17) in
cells treated with VP16 compared with the control
(Fig. 1a). We also showed images from the preliminary
experiment (Fig. 1b) and shared with them the basic trou-
ble shooting ideas. In addition, a ladder pattern typical of
endonucleosomal DNA degradation can be observed when
cells undergo apoptosis (Fig. 2).
Conclusion
The new laboratory curriculum is designed to accomplish
the following student learning objectives: motivate students
for scientific research; prepare students for techniques and
methods they will utilize in later lab classes and integrate
theory and practice to ensure that students understand
why and how each technique is used. Students are
expected to analyze problems, locate relevant material in
library and computer-based resources, and develop inde-
pendent study habits. In addition, students are expected to
assume responsibility for their own learning and to contrib-
ute to the education of their colleagues. As we facilitate the
cultivation of innovative talents, an integrated approach to
technique and research may be just what we need [1].
In addition to the typical course evaluations, 56 stu-
dents were asked to fill out a survey about the course
either several months or a year after taking the course
(Table III) [17]. This timing allowed the students to gauge
what they had learned and determine whether they were
able to maintain the knowledge from the course. All of the
students indicated that the course was beneficial both for
building a foundation of knowledge and for acquiring of
bench experience and training. Also, we designed open
questions for students to get suggestions from them on how
432
Biochemistry and
Molecular Biology Education
to improve the outcome of our course [18]. Typical com-
ments include Some equipment should be updated,
Expecting more than just one such project-oriented
course and Expecting a more challenging process where
we can be involved in the experimental design of solving a
scientific problem through literature learning and then
practicing it. Encouraged by the positive feedback from
students, we still need to work on solving the essential
issues, such as equipment updating and designing more
project-oriented components presented by students. In fact,
we are actively planning and preparing to add a
Functional analysis of genes in cancer biology through
gain of function and loss of function methods component
into the new curriculum, which will involve experiments
such as plasmid/siRNA transfection, real-time polymerase
chain reaction (RT-PCR), phenotype observation, etc.
Finally, the current reform only covers undergraduates in
the eight-year M.D./Ph.D. programs, and we are planning
to expand the accessibility of this course to students of
other training programs in PKUHSC in the coming years.
Acknowledgements
The authors thank Ph.D. student Jianguo Yang (PKUHSC,
Beijing, China) for his kind assistant on the distribution,
collection, and analyses of the questionnaires. Authors are
grateful to senior Ph.D. students David Gallo and Tina Sing
in Dr. Grant Browns lab (Department of Biochemistry, Uni-
versity of Toronto, Canada) for the editorial review of this
paper. This work was supported by Grants (No. J1103605/
J0108, J1030831/J0108) of Fostering Talents in Basic
Research from the National Natural Science Foundation of
China, Grant of Key Project in Education and Teaching
Research from PKUHSC (2014), and a Grant (2012-SY-19 to
X. Yi.) from Chinese Medical Association.
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