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An undergraduate cell biology lab Western Blotting to detect proteins from Drosophila eye

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We have developed an undergraduate laboratory to allow detection and localization of proteins in the compound eye of Drosophila melanogaster, a.k.a fruit fly. This lab was a part of the undergraduate curriculum of the cell biology laboratory course aimed to demonstrate the use of Western Blotting technique to study protein localization in the adult eye of Drosophila. Western blotting, a two-day laboratory exercise, can be used to detect the presence of proteins of interests from total protein isolated from a tissue. The first day involves isolation of proteins from the tissue and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide) gel electrophoresis to separate the denatured proteins in accordance to their molecular weight/s. The separated proteins are then transferred to the Nitrocellulose or Polyvinylidene difluoride (PVDF) membrane in an overnight transfer. The second day lab involves detection of proteins (transferred to the membrane) using Ponceau-S stain, followed by immunochemistry to detect the protein of interest along the total protein transferred to the membrane. The presence of our protein of interest is carried out by using a primary antibody against the protein, followed by binding of secondary antibody which is tagged to an enzyme. The protein band can be detected by using the kit, which provides substrate to the enzyme. The protein levels can be quantified, compared, and analyzed by calculating the respective band intensities. Here, we have used fly eyes to detect the difference in level of expression of Tubulin (Tub) and Wingless (Wg) proteins in the adult eye of Drosophila in our class. The idea of this laboratory exercise is to: (a) familiarize students with the underlying principles of protein chemistry and its application to diverse areas of research, (b) to enable students to get a hands-on-experience of this biochemical technique.
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Teaching Notes Dros. Inf. Serv. 100 (2017)
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York; Law, L.W., 1938, Proc. Natl. Acad. Sci. USA 24: 546-550; Lindsley, D.L., and G.G. Zimm 1992, The
Genome of Drosophila melanogaster. Academic Press Inc., New York; Niklasson, M., H. Petersen, and E.D.
Parker 2000, Pedobiologia 44: 476-488; Pra, D., et al., 2008, Biometals 21: 289-297; Woodruff, R.C., and
J.P. Russell 2011, Dros. Inf. Serv. 94: 184-186.
An undergraduate cell biology lab: Western Blotting to detect proteins from
Drosophila eye.
Gogia, Neha1, Ankita Sarkar1, and Amit Singh1,2,3,4,5. 1Department of Biology, University
of Dayton, Dayton, OH; 2Center for Tissue Regeneration and Engineering at Dayton
(TREND), Dayton, OH; 3Premedical Programs, University of Dayton, Dayton, OH 45469-2320; 4Center for
Genomic Advocacy (TCGA), Indiana State University, Terre Haute, IN; 5Corresponding author email:
asingh1@udayton.edu
Abstract
We have developed an undergraduate laboratory to allow detection and localization of proteins in the
compound eye of Drosophila melanogaster, a.k.a fruit fly. This lab was a part of the undergraduate
curriculum of the cell biology laboratory course aimed to demonstrate the use of Western Blotting technique to
study protein localization in the adult eye of Drosophila. Western blotting, a two-day laboratory exercise, can
be used to detect the presence of proteins of interests from total protein isolated from a tissue. The first day
involves isolation of proteins from the tissue and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide) gel
electrophoresis to separate the denatured proteins in accordance to their molecular weight/s. The separated
proteins are then transferred to the Nitrocellulose or Polyvinylidene difluoride (PVDF) membrane in an
overnight transfer. The second day lab involves detection of proteins (transferred to the membrane) using
Ponceau-S stain, followed by immunochemistry to detect the protein of interest along the total protein
transferred to the membrane. The presence of our protein of interest is carried out by using a primary antibody
against the protein, followed by binding of secondary antibody which is tagged to an enzyme. The protein
band can be detected by using the kit, which provides substrate to the enzyme. The protein levels can be
quantified, compared, and analyzed by calculating the respective band intensities. Here, we have used fly eyes
to detect the difference in level of expression of Tubulin (Tub) and Wingless (Wg) proteins in the adult eye of
Drosophila in our class. The idea of this laboratory exercise is to: (a) familiarize students with the underlying
principles of protein chemistry and its application to diverse areas of research, (b) to enable students to get a
hands-on-experience of this biochemical technique. Keywords: Drosophila melanogaster, eye, Western Blot,
protein estimation. localization of proteins, SDS-PAGE gel electrophoresis.
Introduction
Recent educational research on teaching biology to undergraduates has raised concerns about how
traditional approaches in large classes fail to reach many students and thereby emphasized on the need for
more hand-on experiential learning instructions (Puli and Singh, 2011; Tare et al., 2009; Tare and Singh, 2008;
Uman and Singh, 2011; Wood, 2009; Woodin et al., 2009). One of the hallmarks of the modern day science
education is experiential learning, which allows students to get a hands-on-experience to understand latest
scientific research and concepts. In modern day undergraduate curriculum, research is an important
component of habits of inquiry and learning (Puli and Singh, 2011; Tare et al., 2009; Tare and Singh, 2008;
Uman and Singh, 2011). Efforts have been channeled to develop a repertoire of laboratory courses to expose
undergraduates to modern day biology concepts and techniques used in biomedical research. The new text
books provide exhaustive and detailed information through movies and illustrations on how proteins play a
role in a biological function and what approaches can be used to determine their localization as well as
Dros. Inf. Serv. 100 (2017) Teaching Notes
219
quantitate them using Western Blot approach. Despite the utility of animations and videos the best way of
learning is through hands-on experiential learning (Puli and Singh, 2011; Tare et al., 2009; Tare and Singh,
2008; Wood, 2009). However, it comes with a cost of time and resources. We devised a laboratory to
introduce students to the Western Blot technique, its principle and applications, which will allow students to
determine presence or absence of a protein in a particular tissue and how to semi-quantitatively estimate a
protein in a sample. Furthermore, this exercise can be finished in two laboratory sessions with some
preparation done prior to the demonstration to the students (Figure 1).
Figure 1. Schematic presentation of
time line for Western Blot analysis.
We have developed a two-day
western blot protocol for
undergraduate laboratory course.
This strategy will allow
demonstration of this modern day
technique to undergraduate students.
We have developed this
laboratory exercise to study the
ubiquitously expressed Tubulin
(Tub) and Wingless (Wg) protein in
the adult eye of Drosophila. The
Drosophila model is highly versatile
as it is easy to rear flies in masses in
a short period of time (Puli and Singh, 2011; Singh et al., 2012; Tare et al., 2009; Tare and Singh, 2008).
Furthermore, there are several eye specific mutants available in flies if you want to show comparison of gene
expression among various genetic backgrounds. Drosophila model can be easily used to demonstrate protein
isolation, detection and quantitation.
Western Blotting technique (or immunoblotting) was first described by Towbin et al. (Towbin et al.,
1979). Since then, this technique has become one of the widely used techniques in the field of basic sciences.
Western blot is a highly sensitive biochemical technique, which uses the property of monoclonal/polyclonal
antibodies (highly specific in nature) to bind to their respective antigens. It is mainly used for the detection,
presence/absence, and finding differences in the expression level of a particular protein, or in characterization
of proteins (Kim, 2017). Western blot involves isolation/identification of specific proteins of interest from
tissue samples, or mixture of proteins extracted from cells, which are later quantified, normalized, denatured
(in order to convert their complex structure of protein into its simpler forms). The denatured polypeptides in
the protein sample are then separated on the gel based on their size, molecular weight (Kilo-Daltons (kDa),
using SDS-PAGE (denatured) gel electrophoresis (Kim, 2017; Mahmood and Yang, 2012). The gel
containing the separated protein bands is then placed onto the nitrocellulose or PVDF membrane, and the
protein bands are then electrophoretically transferred from the gel on to the membrane. These membranes are
then subjected first to the blocking step (5% Bovine Serum Albumin, BSA, in order to prevent non-specific
binding of antibodies onto the membrane). After the blocking step, the membranes are treated and incubated
with both primary (specific to the target protein), and secondary antibodies (specific to the primary antibody,
covalently bound/labeled with enzymes). The enzyme becomes active upon availability of its chromogenic
substrate and causes a color reaction. The development of a colored product (using these set of specific
enzyme and substrate reactions) is detected and analyzed using gel documentation (BioSpectrum 500) system.
Teaching Notes Dros. Inf. Serv. 100 (2017)
220
Table 1. List of reagents and solutions used in the Western Blotting lab.
Solutions Volume Composition Preparation/Catalog No.
4X Separation Buffer 500ml Tris Base 90g
Sodium dodecyl sulfate
(SDS) – 2g
Adjust the pH to 8.8 with HCl and make
up the volume to 500ml with autoclaved
water. Store at room temperature.
4X Stacking buffer 500ml Tris Base- 30.25g
Sodium dodecyl sulfate
(SDS) - 2g
Adjust the pH to 6.8 with HCl and make
up the volume to 500ml with autoclaved
water. Store at room temperature.
1X Tris / Glycine / SDS buffer
(Running Buffer)
1Liter 10X Tris/Glycine/SDS
buffer - 100ml
Autoclave Water - 900ml
100ml of 10X Tris/Glycine/SDS buffer is
dissolved in 900 ml autoclaved water.
Store at room temperature.
1X Tris / Glycine buffer
(Transfer buffer)
1Liter 10X Tris/Glycine buffer -
100ml
Methanol - 200ml
Autoclaved water - 700ml
100ml of 10X Tris/Glycine buffer and
200ml of methanol is dissolved in 700ml
of autoclaved water. Store at 4°C.
1X TBST
(TBS Tween-20 buffer)
1Liter 20X TBS Tween-20 buffer
(readymade) - 50ml
Autoclave Water 950ml
50ml of 20X TBS Tween-20 buffer is
dissolved in (Make up the volume with)
950ml autoclaved water. Store at room
temperature.
70% Ethanol 100ml Reagent Alcohol - 70ml
Autoclave Water - 30ml
70ml Reagent Alcohol is dissolved in 30ml
of Autoclave Water and stored at room
temperature.
5% BSA
(Bovine Serum Albumin)
10ml BSA – 0.5g
1X TBS Tween-20 buffer
(TBST) – 10ml
0.5g BSA is dissolved in 10ml of 1XTBST
and stored at 4°C.
10% APS
(Ammonium persulfate)
(freshly prepared)
1ml APS – 0.1g
Autoclave water 1ml
0.1g APS dissolved in 1 ml of autoclave
water in a sterile tube.
TEMED (Tetramethylethyl
enediamine) 20gm
(26ml) Ready to use Fischer Scientific, Cat. #BP150-20
2X Sample Buffer
(Laemmli buffer) Concentrate
1 vial Ready to use SIGMA, Cat. #S3401
Phenylmethanesulfonyl
fluoride (PMSF) 1ml PMSF 0.035g
Isopropanol 1ml
0.035g PMSF is dissolved 1 ml
isopropanol in a sterile tube and stored at
room temperature.
1% Glacial Acetic Acid 100ml Glacial Acetic Acid 1ml
Autoclave Water 99ml
1ml Glacial Acetic Acid is dissolved in 99
ml autoclaved water and stored at room
temperature.
40%Acrylamide /
bisacrylamide (29:1) 1Liter Ready to use Fischer Scientific, Cat. #BP1406-1
Dros. Inf. Serv. 100 (2017) Teaching Notes
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Protocol
The entire methodology of Western Blotting can be divided into four major steps: (1) Sample
preparation, (2) SDS-PAGE gel electrophoresis, (3) Transfer of proteins to the membrane, and (4)
Identification of a protein from a total protein sample using immunochemistry.
DAY I
1. Sample Preparation
We have used Drosophila adult eyes as the tissue source for the total protein isolation. We used fruit
flies as they are easy to rear and large number of flies can be generated in a small time window as life cycle if
just 12 days long at room temperature (Singh et al., 2012; Tare et al., 2013). The biological samples, ~25
adult fly heads are first separated from their respective adult fly bodies using sterilized tweezers and are
collected in labelled tubes that are kept on ice. To each tube, 50 µl of Laemmli 2× Concentrate Sample Buffer,
(SIGMA, Cat.# S3401-1VL) and 3 µl of Phenyl methane sulfonyl fluoride (PMSF) (SIGMA, Cat. #P7626-
5G), a protease inhibitor, is added. The tubes are labelled and the samples are macerated thoroughly with a
sterilized pestle. They are then boiled at 100°C for 10-15 minutes and are immediately kept on ice for 10
minutes. The samples are then subjected to centrifugation for 10 minutes at 10,000 rpm and then snap chilled
on ice again for 10 minutes. The supernatant is transferred into a labelled fresh tube, which is later stored at a
low temperature of -20°C. The total protein concentration in a sample is determined by calculating absorbance
at 280 nm wavelength using spectrophotometer (Nanodrop) along with the control (2× sample buffer can be
used as control. The samples are then normalized by calculating the amount of protein required for a total
concentration of 30 or 40 µg/ml per well and diluting it by adding 2× sample buffer (loading a total volume of
10 µl per well).
Table 2. Recipe for preparation of SDS-PAGE gel. The reagents required for preparation of
a 10% gel are mentioned below.
4X Separating Gel Volume req. for
preparing 1 gel Volume req. for
preparing 2 gels
40% Acryalmide/bisacrylamide (29:1) 1.25 ml 2.5 ml
4X separation buffer 1.25 ml 2.5 ml
Autoclaved water 2.5 ml 5 ml
10%APS (freshly prepared, stored at 4°C) 50 µl 100 µl
TEMED (stored at 4°C) 5 µl 10 µl
4X Stacking Gel
Volume req. for
preparing 1 gel
Volume req. for
preparing 2 gels
40% Acryalmide/bisacrylamide (29:1) 0.25 ml 0.5 ml
4X stacking buffer 0.625 ml 1.25 ml
Autoclaved water 1.625 ml 3.25 ml
10%APS (freshly prepared, stored at 4°C) 25 µl 50 µl
TEMED (stored at 4°C) 2.5 µl 5 µl
2. SDS-PAGE Gel electrophoresis
In order to save time, we sometimes use precast gels (Mini Protean stain free precast gels from Bio-
Rad). For casting gels, the two glass plates are washed first with autoclaved water and cleaned with 70%
Teaching Notes Dros. Inf. Serv. 100 (2017)
222
ethanol (Reagent Alcohol, Fisher Scientific, Cat. #A962-4) for setting them up in the gel apparatus. The
polyacrylamide gels are formed by polymerization of acrylamide and bis-acrylamide (bis, N,N’-methylene-
bis-acrylamide, Fischer Scientific, Cat. #BP1406-1). Polymerization is initiated by Ammonium Persulfate
(APS) (Fisher Scientific, Cat. #BP179-100) and TEMED (Tetramethylethylenediamine) (Fisher Scientific,
Cat. #BP150-20). TEMED accelerates the rate of formation of free radicals from persulfate and these in turn
catalyze polymerization. Therefore, the 4× separating gel mixture is prepared first and mixed well before
addition of both APS and TEMED. APS is added first followed by addition of TEMED (Table 2). The gel
components are mixed thoroughly to ensure homogenous solution. The gel mixture (without any further
delay) is poured inside the space (up to 70% of total size of glass plate) present between the two glass plates.
Around 350 µl of 70% ethanol (or just needed enough to cover the surface) is poured on top of the
polymerizing gel to prevent the gel from coming in contact with air, which may trigger rapid polymerization of
only the upper part of the gel. After the gel has polymerized (after ~ 35 minutes), the 70% ethanol is poured
out, and the top of the gel is washed thoroughly with autoclaved water.
The 4× stacking gel mixture is prepared in a similar fashion as 4× separating gel mixture, and the
volume and concentration of chemicals required to prepare a 4× stacking gel is mentioned in Table 2. Once
prepared, 4× stacking gel mixture is poured on top of the polymerized 4× separating gel using a micropipette.
The combs are inserted slowly just to make sure no bubbles are trapped inside and the gel is left undisturbed to
complete the polymerization process. Once the gel has completely polymerized, the gel plates are fitted inside
(lower glass plate facing inside) the gel cassette (containing red-positive and black-negative electrodes). The
gel cassette is then lodged inside the electrophoresis unit that contains 1× Tris/Glycine/SDS (1× TG-SDS)
(10× Tris/Glycine/SDS Buffer, BIO-RAD, Cat. #161-0732) buffer (Table 1). The top of the gel cassette unit is
also filled with the 1× TG-SDS buffer, which is required to complete the circuit. The combs are then taken out
and wells are washed nicely with 1× TG-SDS buffer (to remove any loose pieces of acrylamide, which if left
untreated, can block the wells during the gel run). The normalized protein samples are mixed with 2×
Laemmli Concentrate Sample Buffer to make up the total volume to 10 µl, which is then loaded into the
respective wells of the gel. A molecular weight marker (Precision Plus Protein Standards Kaleidoscope (BIO-
RAD, Cat. #161-0375) is loaded (~4.5 µl) adjacent to the experimental samples in order to get an idea about
the size or molecular weight of the protein of interest (Kim, 2017; Mahmood and Yang, 2012; Weber and
Osborn, 1969), which is measured in Kilo-Daltons (kDa). The gel is then subjected to electrophoresis using
power supply unit at 90V for 2.5-3 hours.
3. Transfer of proteins to the membrane
The 1× Tris Glycine buffer (or TG, transfer buffer) (10× Tris/Glycine Buffer, BIO-RAD, Cat. #161-
0734) is prepared according to Table 1 and is kept at 4°C for pre-cooling. Both Nitrocellulose or PVDF
membranes (Immun-Blot PVDF: BIO-RAD, Cat. #162-0177) can be used during the transfer process, but
PVDF membranes are more durable, hydrophobic, chemically more inert (as compared to nitrocellulose
membranes), which increases their potential to bind more to protein (Bass et al., 2017). The PVDF membrane
is cut to the size of the gel and is soaked in methanol for 5-10 mins. The membrane, 2 filter papers, 2 sponges
are then transferred into 1× Tris/Glycine buffer (Table 1) in order to equilibrate them before the transfer
process. The glass plates (containing polymerized gel) are taken out from the gel cassette. The upper plate is
removed slowly followed by removal of stacking gel gently from the rest of the gel and the gel is poured with
1× Tris/Glycine buffer to equilibrate.
Preparation of transfer sandwich (to be carried out overnight): It is performed in a tray containing 1×
Tris/Glycine (transfer buffer, Table 1). The sequence for sandwich formation is as follows - The black side of
the sandwich apparatus is placed down in the tray (containing 1× transfer buffer), followed by a sponge
(wetted in 1× transfer buffer) and a rectangular piece of white filter paper (wetted in 1× transfer buffer). The
gel is placed and is covered by placing the nitrocellulose or PVDF membrane onto the gel (make sure no
bubbles are trapped inside). The bubbles are removed by rolling a glass rod on the gel and membrane. Onto
the gel and the membrane, another piece of white filter paper (wetted in 1× transfer buffer) is placed, followed
Dros. Inf. Serv. 100 (2017) Teaching Notes
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by another sponge (wetted in 1× transfer buffer). The sandwich is locked afterwards. The sandwich is then
placed hinge down, with its black side towards the black side (cathode-negative) of the transfer apparatus. The
transfer apparatus is filled with a small ice pack and pre-cooled 1× transfer buffer filled up to the brim. The
transfer process is performed in a cold room at 25 Volts (V), at 4°C for overnight or at 60V, 4°C for 2 hours.
DAY II
After the transfer process is done, the membrane is carefully taken out from the transfer
electrophoresis unit and washed three times with autoclaved water (2 minutes each). The membrane is then
treated two times with 1% Glacial acetic acid solution (ARISTAR, Cat# BDH3094-2.5LG) for 5 minutes each.
The membrane is then stained with Ponceu-S staining solution (SIGMA, CAT. #P7170-1L), while shaking for
5-10 minutes, and is further de-stained with 1% Glacial acetic acid (protein bands are clearly visible at this
stage). Ponceau-S stain marks the protein bands. However, if the bands are not clearly visible, it doesn’t
always mean that it won’t show any signal during the developing process, because West Dura developing kit is
a lot more sensitive than Ponceau-S stain. It can detect approximately 100 ng of protein per band (Ness et al.,
2015). The membrane is then washed three times with autoclaved water (10 minutes each) and is then
equilibrated with 1× TBST (20× TBS Tween-20 Buffer, Thermo Scientific, Cat. #28360) solution three times
(10 minutes each).
4. Identification of a protein from a total protein sample using immunochemistry
Primary and Secondary Antibodies: The membrane is first blocked with 5% Bovine Serum Albumin (BSA)
(Fisher Scientific, Cat. # BP1600-100) prepared in 1× TBST for 1 hour at room temperature and is then
incubated with primary antibody-Monoclonal Anti-α-Tubulin antibody (1:12000) produced in mouse (SIGMA,
Cat. # T5168), Monoclonal Anti-Wingless (1:500) produced in mouse (DSHB, 4D4) prepared in 5% BSA, 1×
TBST, overnight at 4°C or 3-4 hours at room temperature (depending on the time available to instructor). The
membrane is then washed three times with 1× TBST (10 minutes each, more or less number of washes
depends on the antibody used) and is further treated with secondary antibody (Goat anti-Mouse IgG-HRP
1:5000) (Santa Cruz Biotechnology, Cat. Sc-2005) prepared in 5% BSA, 1× TBST for 30 minutes at room
temperature or for 2 hours at 4°C. After secondary antibody treatment, the membrane is washed three times
with 1× TBST, 10 minutes each to remove any extra unbound antibody left on the membrane to avoid
nonspecific signal.
Developing protein bands and detection: The Super Signal West Dura Extended Duration Substrate Kit
(Thermo Scientific, Cat. #34076) (highly sensitive in nature) is used for developing the protein bands. The kit
allows detection of even mid-femto gram of antigen by oxidizing luminol based chemiluminescent substrate
for Horseradish peroxidase (HRP) detection. Equal volumes of SuperSignal West Dura Stable Peroxide
Buffer (Prod. # 1859025) and SuperSignal West Dura Luminol/Enhancer Solution (Prod. #1859024) (~1 ml)
are mixed together in a tube to form the developing solution and is applied on to the membrane. The
membrane is then shaken manually, just to make sure the developing solution covers the entire surface of the
membrane. The membrane is incubated with developing solution for 5 minutes at room temperature and the
solution is drained afterwards. The membrane is then analyzed and imaged in a Gel Documentation System
(UVP BioSpectrum 500 Imaging System with LM-26 and BioChemi 500 Camera f/1.2, S/N021110-001) with
exposure time (limit range from 5 sec to 1 minute, and longer if necessary).
Advantages of using Western blotting technique
1. One of the challenges of teaching a laboratory course is the willingness of the institution to invest in setting
up the lab. Therefore, the use of cost- and time-effective exercises can facilitate easy implementation of these
laboratory programs. The solutions used for the Western Blot analysis are commercially available and are
inexpensive.
Teaching Notes Dros. Inf. Serv. 100 (2017)
224
Figure 2. Western Blot Analysis to detect proteins. Total protein sample
isolated from wild-type adult eye of Drosophila were separated by SDS-
PAGE electrophoresis. The (A) Tubulin and (B) Wingless protein were
detected using the Tubulin and Wingless antibody. (A) A band
corresponding to 55 kDa molecular weight, was detected which
corresponds to Tubulin. (B) A band corresponding to 46 kDa molecular
weight, corresponding to Wingless (Wg) was detected. Images were
captured using the BioSpectrum® 500 Imaging System. The same blot
was first used to detect Tubulin. It was then stripped and used for
detecting Wingless protein.
2. It is challenging for undergraduate students to learn these technique
from books, animations, or tutorials. This teaching note will help develop
experiential learning opportunities for students and learn this technique in
an easy and effective manner.
3. The first step of Western blot involves separation of total proteins
using SDS page electrophoresis. Therefore, this lab can be coupled with
the SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) laboratory.
Thus, in two days you can demonstrate two techniques as it just adds one more lab for identification of a
protein using immunohistochemistry (as transfer of proteins from gel to membrane can be done overnight).
Moreover, the procedure has incubation steps. The time between the incubations can be utilized for interaction
with lab instructor, clarifying the concepts and class discussions.
4. The students get general overview of Western Blotting technique, which is highly sensitive and can detect
as little as 0.1ng of protein. This exercise provides hands-on experience of this technique starting from sample
preparation to visualizing the proteins onto the membrane.
5. The technique employs use of antigen-antibody reactions (highly specific in nature) and thus has the
capability of detecting the protein of interest even from a mixture or a solution containing 300,000 diverse
range of proteins. It can also help detect the immunogenic responses (caused by bacteria or viruses), or can be
used to study regulation of genes known to cause asthma, allergy (García-Sánchez and Marqués-García, 2016),
or for detection and diagnosis of deadly diseases like human immunodeficiency virus (HIV) (Feng et al.,
2017).
6. This Western blot analysis utilizes standard protein chemistry and is easy to demonstrate in a undergraduate
laboratory setup as it does not need educational demonstration kits that minimize the exposure of students to
details. It will add to the skill set of students and will help develop a core of trained individuals suitable for
academics or industrial settings.
Acknowledgments: This laboratory exercise was designed in the Department of Biology, at the
University of Dayton. NG, AS are supported by graduate program of the University of Dayton. AS is
supported by NIH1R15GM124654-01 and Stem Catalyst Grant.
References: Bass, J.J., D.J. Wilkinson, D. Rankin, B.E. Phillips, N.J. Szewczyk, K. Smith, and P.J.
Atherton 2017, Scand. J. Med. Sci. Sports. 27: 4-25; Feng, X., J. Wang, Z. Gao, Y. Tian, L. Zhang, H. Chen,
T. Zhang, L. Xiao, J. Yao, W. Xing, et al., 2017, J. Clin. Virol. 88: 8-11; García-Sánchez, A., and F. Marqués-
García 2016, Review of Methods to Study Gene Expression Regulation Applied to Asthma. In: Molecular
Genetics of Asthma (Isidoro García, M., ed.), pp. 71-89. New York, NY: Springer New York; Kim, B., 2017,
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Methods Mol. Biol. 1606: 133-139; Mahmood, T., and P.C. Yang 2012, N. Am. J. Med. Sci. 4: 429-434;
Ness, T.L., R.L. Robinson, W. Mojadedi, L. Peavy, and M.H. Weiland 2015, Biochem. Mol. Biol. Educ. 43:
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Mechanisms of Axial Patterning: Mechanistic Insights into Generation of Axes in the Developing Eye. In:
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Real time quantitative PCR to demonstrate gene expression in an
undergraduate lab.
Mehta, Abijeet Singh1, and Amit Singh1,2,3,4,5. 1Department of Biology, University of
Dayton, Dayton, OH; 2Center for Tissue Regeneration and Engineering at Dayton
(TREND), Dayton, OH; 3 Premedical Programs, University of Dayton, Dayton, OH 45469-
2320; 4Center for Genomic Advocacy (TCGA), Indiana State University, Terre Haute, IN. Corresponding
Author Email: asingh1@udayton.edu
Abstract
The objective of this teaching note is to develop a laboratory exercise, which allows students to get a
hands-on experience of a molecular biology technique to analyze gene expression. The short duration of the
biology laboratory for an undergraduate curriculum is the biggest challenge with the development of new labs.
An important part of cell biology or molecular biology undergraduate curriculum is to study gene expression.
There are many labs to study gene expression in qualitative manner. The commonly used reporter gene
expression studies are primarily qualitative. However, there is no hands-on experience exercise to
quantitatively determine gene expression. Therefore, it is necessary to design a laboratory exercise that
enables the students to carry out cell or molecular biological assays in the desired time. Here we report a
laboratory where we can introduce students to gene expression using the real time Quantitative Polymerase
Chain Reaction (RT-qPCR) by comparative CT method to analyze expression of genes in Drosophila tissues.
Keywords: Drosophila melanogaster, eye, real time quantitative PCR, gene expression.
Introduction
A challenging situation emerging with fast paced growth on the research front in various disciplines of
Biology is to introduce emerging new concepts into the undergraduate curriculum too (Puli and Singh, 2011;
Tare et al., 2009; Tare and Singh, 2008; Usman and Singh, 2011; Wood, 2009). Interestingly, central dogma
of molecular biology is an age old and time-tested concept that has been delivered in the undergraduate
classroom. Even though the basic concept about central dogma is that genetic information of an organism or a
cell is stored in nucleic acid DNA, which is then transcribed into single stranded RNA, and finally translated to
protein but the strategies to study gene expression (qualitatively and quantitatively) have been evolving to
date. The conventionally used approaches to deliver this curriculum in laboratory class are to use reporter
gene expression, immunohistochemistry, or using protein trap lines. However, the majority of these
techniques are qualitative, or to some extent semi-quantitative, in nature. Therefore, there are not many
quantitative approaches to determine or compare levels of gene expression among different tissues that can be
used for classroom demonstration.
... The protein samples were extracted from heads of Wild-type, GMR > FUS, GMR > FUS-R518K, and GMR > FUS-R521C adult flies using a standardized protocol (Gogia et al., 2017). The primary antibodies used were Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) (1:3000, Cell Signaling) and anti-FUS (1:1500, A300-302A, Bethyl laboratories). ...
... We decided to test if Hippo signaling downregulation rescues FUS mediated neurodegeneration by blocking cell death. We used TUNEL staining, which marks the fragmented DNA, to mark the nuclei of the dying neurons (Cutler et al., 2015;Gogia et al., 2017;McCall and Peterson, 2004;Tare et al., 2011;White et al., 1994). We performed TUNEL staining in third instar larval eye-antennal imaginal discs of the wild-type larvae (Fig. 4A), GMR > FUS (Fig. 4B). ...
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Amyotrophic Lateral Sclerosis (ALS), a late-onset neurodegenerative disorder characterized by the loss of motor neurons in the central nervous system, has no known cure to-date. Disease causing mutations in human Fused in Sarcoma (FUS) leads to aggressive and juvenile onset of ALS. FUS is a well-conserved protein across different species, which plays a crucial role in regulating different aspects of RNA metabolism. Targeted misexpression of FUS in Drosophila model recapitulates several interesting phenotypes relevant to ALS including cytoplasmic mislocalization, defects at the neuromuscular junction and motor dysfunction. We screened for the genetic modifiers of human FUS-mediated neurodegenerative phenotype using molecularly defined deficiencies. We identified hippo (hpo), a component of the evolutionarily conserved Hippo growth regulatory pathway, as a genetic modifier of FUS mediated neurodegeneration. Gain-of-function of hpo triggers cell death whereas its loss-of-function promotes cell proliferation. Downregulation of the Hippo signaling pathway, using mutants of Hippo signaling, exhibit rescue of FUS-mediated neurodegeneration in the Drosophila eye, as evident from reduction in the number of TUNEL positive nuclei as well as rescue of axonal targeting from the retina to the brain. The Hippo pathway activates c-Jun amino-terminal (NH2) Kinase (JNK) mediated cell death. We found that downregulation of JNK signaling is sufficient to rescue FUS-mediated neurodegeneration in the Drosophila eye. Our study elucidates that Hippo signaling and JNK signaling are activated in response to FUS accumulation to induce neurodegeneration. These studies will shed light on the genetic mechanism involved in neurodegeneration observed in ALS and other associated disorders.
... Furthermore, differences in methods across labs, inconsistent handling of samples, nonuniform binding of dye, among other issues, lead to inaccurate and irreproducible results. Other techniques such as semiquantitative traditional/quantitative Western blots can only study levels of protein expression that cannot be performed spatially in real time [25]. Use of dye-based assays to detect ROS are highly popular but relatively qualitative. ...
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Numerous imaging modules are utilized to study changes that occur during cellular processes. Besides qualitative (immunohistochemical) or semiquantitative (Western blot) approaches, direct quantitation method(s) for detecting and analyzing signal intensities for disease(s) biomarkers are lacking. Thus, there is a need to develop method(s) to quantitate specific signals and eliminate noise during live tissue imaging. An increase in reactive oxygen species (ROS) such as superoxide (O 2 • ⁻ ) radicals results in oxidative damage of biomolecules, which leads to oxidative stress. This can be detected by dihydroethidium staining in live tissue(s), which does not rely on fixation and helps prevent stress on tissues. However, the signal-to-noise ratio is reduced in live tissue staining. We employ the Drosophila eye model of Alzheimer's disease as a proof of concept to quantitate ROS in live tissue by adapting an unbiased method. The method presented here has a potential application for other live tissue fluorescent images.
... Protein samples were prepared from (n = ∼50) adult eyes from Canton-S (wild-type), GMR> Aβ42, GMR> Aβ42+hpo following standardized protocols (Gogia et al., 2017). The samples were loaded in the following sequence: Lane 1-Molecular weight marker (BIORAD Precision Plus Protein Kaleidoscope Prestained Catalog Number #1610375), Lane 2-Wild-type (Canton-S), Lane 3-GMR> Aβ42, Lane 4-GMR> Aβ42+hpo (gain-of-function), Lane 5-GMR> Aβ42+hpo RNAi (loss-offunction). ...
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Alzheimer's disease (AD, OMIM: 104300) is an age-related disorder that affects millions of people. One of the underlying causes of AD is generation of hydrophobic amyloid-beta 42 (Aβ42) peptides that accumulate to form amyloid plaques. These plaques induce oxidative stress and aberrant signaling, which result in the death of neurons and other pathologies linked to neurodegeneration. We have developed a Drosophila eye model of AD by targeted misexpression of human Aβ42 in the differentiating retinal neurons, where an accumulation of Aβ42 triggers a characteristic neurodegenerative phenotype. In a forward deficiency screen to look for genetic modifiers, we identified a molecularly defined deficiency, which suppresses Aβ42-mediated neurodegeneration. This deficiency uncovers hippo (hpo) gene, a member of evolutionarily conserved Hippo signaling pathway that regulates growth. Activation of Hippo signaling causes cell death, whereas downregulation of Hippo signaling triggers cell proliferation. We found that Hippo signaling is activated in Aβ42-mediated neurodegeneration. Downregulation of Hippo signaling rescues the Aβ42-mediated neurodegeneration, whereas upregulation of Hippo signaling enhances the Aβ42-mediated neurodegeneration phenotypes. It is known that c-Jun-amino-terminal kinase (JNK) signaling pathway is upregulated in AD. We found that activation of JNK signaling enhances the Aβ42-mediated neurodegeneration, whereas downregulation of JNK signaling rescues the Aβ42-mediated neurodegeneration. We tested the nature of interactions between Hippo signaling and JNK signaling in Aβ42-mediated neurodegeneration using genetic epistasis approach. Our data suggest that Hippo signaling and JNK signaling, two independent signaling pathways, act synergistically upon accumulation of Aβ42 plaques to trigger cell death. Our studies demonstrate a novel role of Hippo signaling pathway in Aβ42-mediated neurodegeneration.
... Western Blot. Protein sample were prepared from third instar eye imaginal disc from Wild type, GMR > Aβ42, GMR > Aβ42 + Lun larvae following the standardized protocol 27,93 . The Phospho SAPK/JNK (Cell Signaling Thr183/Tyr185) (81E11) Rabbit antibody was used at 1:1000 dilution. ...
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Alzheimer's disease (AD), a fatal progressive neurodegenerative disorder, also results from accumulation of amyloid-beta 42 (Aβ42) plaques. These Aβ42 plaques trigger oxidative stress, abnormal signaling, which results in neuronal death by unknown mechanism(s). We misexpress high levels of human Aβ42 in the differentiating retinal neurons of the Drosophila eye, which results in the Alzheimer's like neuropathology. Using our transgenic model, we tested a soy-derived protein Lunasin (Lun) for a possible role in rescuing neurodegeneration in retinal neurons. Lunasin is known to have anti-cancer effect and reduces stress and inflammation. We show that misexpression of Lunasin by transgenic approach can rescue Aβ42 mediated neurodegeneration by blocking cell death in retinal neurons, and results in restoration of axonal targeting from retina to brain. Misexpression of Lunasin downregulates the highly conserved cJun-N-terminal Kinase (JNK) signaling pathway. Activation of JNK signaling can prevent neuroprotective role of Lunasin in Aβ42 mediated neurodegeneration. This neuroprotective function of Lunasin is not dependent on retinal determination gene cascade in the Drosophila eye, and is independent of Wingless (Wg) and Decapentaplegic (Dpp) signaling pathways. Furthermore, Lunasin can significantly reduce mortality rate caused by misexpression of human Aβ42 in flies. Our studies identified the novel neuroprotective role of Lunasin peptide, a potential therapeutic agent that can ameliorate Aβ42 mediated neurodegeneration by downregulating JNK signaling.
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Newts utilize their unique genes to restore missing parts by strategic regulation of conserved signaling pathways. Lack of genetic tools pose challenges to determine the function of such genes. Therefore, we used the Drosophila eye model to demonstrate the potential of 5 unique newt (Notophthalmus viridescens) gene(s), viropana1-viropana5 (vna1-vna5), which were ectopically expressed in L² mutant and GMR-hid, GMR-GAL4 eye. L² exhibits the loss of ventral half of early eye and head involution defective (hid) triggers cell-death during later eye development. Surprisingly newt genes significantly restore missing photoreceptor cells both in L² and GMR>hid background by upregulating cell-proliferation and blocking cell-death, regulating evolutionarily conserved Wingless (Wg)/Wnt signaling pathway and exhibit non-cell-autonomous rescues. Further, Wg/Wnt signaling acts downstream of newt genes. Our data highlights that unique newt proteins can regulate conserved pathways to trigger a robust restoration of missing photoreceptor cells in Drosophila eye model with weak restoration capability.
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All multicellular organisms require axial patterning to transform a single-layer organ primordium to a three-dimensional organ. It involves delineation of anteroposterior (AP), dorsoventral (DV), and proximodistal (PD) axes. Any deviation in this fundamental process results in patterning and growth defects during organogenesis. The Drosophila eye is an excellent model to study axial patterning. In the Drosophila eye, DV lineage is the first axis to be determined, which is followed by generation of the AP axis. The default state of the Drosophila early eye primordium is ventral, and the dorsal fate is established by onset of expression of dorsal eye fate selector pannier (pnr)in a group of cells on the dorsal eye margin. The boundary between dorsal and ventral compartments is the site for activation of Notch (N) signaling and is referred to as the equator. Activation of N signaling is crucial for initiating the cell proliferation and differentiation in the developing Drosophila eye imaginal disc. This chapter will focus on (a) how axial patterning occurs in the developing Drosophila eye, (b) how the developing eye field gets divided into dorsal and ventral cell populations, and (c) how DV patterning genes contribute toward the growth and patterning of the fly retina. © 2013 Springer Science+Business Media New York. All rights are reserved.
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