Separation of protein by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and

detection of protein expression by Western blotting

Aim:
To separate the total protein isolated from human cell line MDA-MB-231 (Breast cancer cell
line) and HEK-293 (Human embryonic kidney cell line) by SDS-PAGE and detects the
expression of β-actin by western blot
Principle:
Western blotting or immunoblotting is a method used for identifying a specific protein in a
complex mixture along with determination of its molecular weight. Protein samples are first
electrophoresed on SDS-PAGE. In this process proteins migrate through the gel and they are
separated according to their size and charge. These separated proteins are electro transferred
onto nitrocellulose/PVDF membrane for further analysis. To detect the protein (antigen)
blotted on the membrane it is incubated with an antibody (primary) specific for the protein of
interest. The membrane is then incubated with a second antibody (secondary) which is
specific for the first antibody. The secondary antibodies are covalently attached to an
enzyme, e.g. alkaline phosphatase or horseradish peroxidase. These enzymes form a colored
precipitate upon reacting with a chromogenic substrate. As a result, a visible band can be
seen on the membrane where the primary antibody is bound to the protein. The entire
procedure can be divided into following steps:
SDS-PAGE
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a technique for
separating proteins based on their ability to move within an electrical current, which is a
function of the length of their polypeptide chains or of their molecular weight. This is
achieved by adding SDS detergent to remove secondary and tertiary protein structures and to
maintain the proteins as polypeptide chains. The SDS coats the proteins, mostly proportional
to their molecular weight, and confers the same negative electrical charge across all proteins
in the sample. The most widely used gel system for separating a broad range of proteins by
SDS-PAGE is the Laemmli system (1970) which uses tris-glycine gels comprised of a
stacking gel component and the resolving gel where varying acrylamide gel percentages are
used to separate the proteins based on their mass weight. This classic system uses a
discontinuous buffer system where the pH and ionic strength of the buffer used for running
the gel is different from the buffers used in the stacking gel (Tris-HCl, pH 6.8) and resolving
gel (Tris-HCl, pH 8.8).
The sieving properties of polyacrylamide gels are established by the three-dimensional
network of fibres and pores which is formed as the bifunctionalbisacrylamide cross-links
adjacent polyacrylamide chains (Figure 1). TEMED (N,N,N,N'-tetramethylenediamine)
catalyzes the polymerization reaction by promoting the production of free radicals by
ammonium per sulfate (APS). The effective pore size of gels decreases with the increase of
acrylamide concentration. As the pore size of a gel decreases, the migration rate of protein
through the gel decreases.

Figure 1: Polymerization and crosslinking of acrylamide.

Western blotting
Immunoblotting or Western blotting is the electro transfer of resolved proteins from the
polyacrylamide gel to the nitrocellulose/PVDF membrane in presence of a specific buffer
called transfer buffer. For this transfer procedure, the gel is placed on the membrane and both
of them are sandwiched between two filter papers as shown in Figure 2.

Figure 2: western blot transfer set up

This set is placed between two sponge pads and then placed in a plastic cassette. The entire
set is then placed inside a gel tank filled with cold transfer buffer. The resolved proteins are
transferred to the corresponding positions on the membrane after the electro transfer. The
protein of interest is then immunodetected on the membrane. A suitable blocking reagent
(non-fat dry milk) is used to block the unoccupied sites on the membrane. Then the
membrane is probed with a primary antibody specific to the protein of interest. The primary
antibody binds to the protein (antigen) and an antigen (Ag)-antibody (Ab) complex is formed
on the membrane. The membrane is washed to remove excess unbound primary antibody. It
is then treated with an enzyme-labeled (Alkaline phosphatase/Horseradish peroxidase)
secondary antibody which attaches to the primary antibody of the Ag-Ab complex. Finally,
the membrane is incubated in a solution containing phosphatase or peroxidase substrate
which results in a visible colored band on the membrane where the Ag-Ab complex is
formed. As a result, the molecular weight of the protein of interest can be determined.
Materials required:
Glassware: Measuring cylinder, Beakers, Conical flask.
Others: Cold centrifuge, Protein Electrophoresis apparatus, Blotting Apparatus, Power pack,
Gel rocker, Micropipettes, Tips, Hotplate.
Preparation of reagent:
Cell lysis buffer 30% Acrylamide-bisacrylamide Solution
Tris-HCl, pH 8 = 10 mM Acrylamide = 15 g
EDTA = 1 mM Bis-acrylamide = 0.4 g
EGTA = 0.5mM Volume was made up to 50 ml, filter
Triton X-100 = 1% sterilized and stored at 4°C in amber
SDS = 0.1% bottle
Sodium deoxycholate = 0.1%
Nacl = 140 mM
4x Laemmli sample buffer
Tris-HCl (pH 6.8) = 250 mM
SDS = 8%
Glycerol = 40%
Bromophenol blue = 0.04%
Laemmli buffer is stored at 4°C. Before mixing with protein samples, 100 μl of β-
marcaptoethanol was added to 900 μl Laemmli buffer.
1.5 M Tris-HCl (pH 8.8)
9.08 g of Tris was mixed with 30 ml autoclaved water and pH was set to 8.8 using HCl. The
volume was made up to 50 ml, filter sterilized and stored at RT.
0.5 M Tris-HCl (pH 6.8)
3 g of Tris was mixed with 30 ml autoclaved water and pH was set to 6.8 using HCl. The
volume was made up to 50 ml, filter sterilized and stored at RT.
10% APS Solution
0.5 g of Ammonium persulphate was mixed with 5 ml of autoclaved water and stored at 4°C.
10x gel running buffer
Tris = 7.5 g
Glycine = 36 g
SDS = 2.5 g
Mixed in autoclaved water, volume was made up to 250 ml and stored at RT. Before use,
buffer was diluted to 1x by adding 100 ml of 10x buffer to 900 ml of autoclaved water.
10x transfer buffer
Tris = 29 g
Glycine = 14.5 g
Mixed in autoclaved water, volume was made up to 250 ml and stored at RT. Before use,
buffer was diluted to 1x by adding 100 ml of 10x buffer and 200 ml of methanol to 700 ml of
autoclaved water.
10x Tris buffer saline (TBS)
Tris = 1.21 g
NaCl = 0.4 g
Mixed with 30 ml autoclaved water and pH was set to 7.6. The volume was made up to 50 ml
and stored at RT.
1x TBST (TBS-Tween-20)

20 ml of 10x TBS was mixed with 180 ml of autoclaved water and 200 μl of Tween-20 was
added to it.
Ponceau S dye solution
Ponceau S powder = 0.1 g
Glacial acetic acid = 5 ml
Distilled water = 95 ml
Blocking solution (10%)
2 g of skimmed milk powder was mixed with 20 ml of 1x TBST.
Antibody dilution solution
1 g of bovine serum albumin (BSA) was mixed with 20 ml of 1x TBST and stored at 4°C.
Procedure
Day 1: Isolation of protein and SDS-PAGE
1. After collecting cells in microcentrifuge tube, 300 μl cell lysis buffer was added and put at
ice for 30 min. Following lysis, cells were centrifuged at 10,000 rpm for 30 minutes.
Supernatants were taken in a fresh microcentrifuge tube and the protein concentration was
measured by Bradford method.
2. Preparation of the resolving gel: In a 50 ml falcon, 10 ml of solution containing the desired
concentration was prepared for 10% resolving gel using the values given below. The
ammonium persulfate solution and TEMED was added finally as indicated below. The
solution was mixed and immediately poured into the gap between the two glass plates. The
gel was kept at room temperature for 30 min to allow polymerization. Immediately after the
gel is poured, methanol was to level the gel.
Resolving gel (10%) - 10 ml
30% Acrylamide-bisacrylamide Solution: 3.4 ml
Distilled water: 3.8 ml
1.5M Tris (pH 8.8): 2.6 ml
10% SDS: 0.1 ml
10% APS: 0.1 ml
TEMED: 0.01 ml
3. After 30 min methanol was poured off by inverting the casting assembly.
4. Preparation of the stacking gel: In a 15 ml falcon, the appropriate volume of solution
containing the desired concentration was prepared for the 4% stacking gel using the values
given below. The ammonium persulfate solution and TEMED was added finally as indicated
below. The solution was mixed and poured immediately on top of the resolving gel and the
comb was placed to avoid air bubbles. It was allowed to solidify for 30 minutes.
Stacking gel (4%) - 5 ml
30% Acrylamide-bisacrylamide Solution: 0.67 ml
Distilled water: 2.975 ml
0.5M Tris (pH 6.8): 1.25 ml
10% SDS: 0.05 ml
10% APS: 0.05 ml
TEMED: 0.005 ml
5. 1x Running Buffer was poured in the unit such that the buffer connects the two electrodes,
and hence completes the flow of current. The comb was removed from the stacking gel
carefully.
6. After determining the concentration, 20 μg of protein from each samples were taken in
microcentrifuge tube containing 4x Laemmli sample buffer. The sample buffer and protein
were mixed at 3:1 ratio and boiled at 100°C in a boiling water bath for 5 min.
7. Samples were loaded in wells as follow:
Lane 1: Protein Sample (MDA-MB-231) – 2.6 μl
Lane 2: Protein Sample (MDA-MB-231) – 2.6 μl
Lane 3: Protein Sample (HEK-293) – 1.8 μl
Lane 4: Protein Sample (HEK-293) – 1.8 μl
Lane 5: Prestained Protein Ladder – 4 μl
8. The power cord was connected to the electrophoretic power supply according to the
conventions: Red - Anode and Black - Cathode. Protein samples were electrophoresed at 90
volts and 100 mA until dye front reaches below of the resolving gel.
9. The gel was carefully removed from in-between the plates using gel cutter and put into the
plastic tray to proceed for western blotting.
10. The gel was assembled with nitrocellulose membrane and filter papers as shown in figure
2. This blotting sandwich was placed within the blotting cassette. Air bubble between gel and
nitrocellulose membrane was removed by using a roller.
11. This cassette was then inserted into the gel transfer apparatus filled with cold 1x transfer
buffer and then connected the transfer unit to power supply as per conventions. Transfer was
carried out at 100V, 400 mA for 1.5 hours.
12. The nitrocellulose membrane was removed after electrophoresis from the blotting cassette
and placed the membrane (with protein side up) in Ponceau S dye taken in a plastic container
to further confirm whether protein samples were transferred to the membrane (figure 3).
After confirmation, dye was removed from membrane by washing in transfer membrane for
2-3 min.
13. The membrane was then placed in 10 ml blocking solution (10% skimmed milk in 1x
TBST) for 2 hours.
14. Following blocking, blocking solution was discarded and the membrane was immersed in
10 ml primary antibody solution which was prepared by adding 1 μl of rabbit anti-human β-
actin antibody into 10 ml antibody dilution solution (5% BSA in 1x TBST). The membrane
was put in the primary antibody solution and mixed gently on a gel rocker for overnight at
4°C.
Day 2: Immunodetection
1. After overnight incubation, primary antibody solution was discarded and the membrane
was washed vigorously with wash buffer (1x TBST) on a gel rocker (at high speed) for 5
min. Washing was repeated for 5 times and the buffer was discarded every time.
2. The membrane was then immersed in 10 ml secondary antibody solution which was
prepared by adding 2 μl of ALP labelled goat anti-rabbit IgG into 10 ml antibody dilution
solution (1x TBST). The membrane was put in the secondary antibody solution and mixed
gently on a gel rocker for 2 hr at RT.
3. After incubation with secondary antibody the membrane was washed vigorously with wash
buffer (1x TBST) on a gel rocker (at high speed) for 5 min. Washing was repeated for 5 times
and the buffer was discarded every time.
4. After washing, the membrane was immersed in 10 ml NBT/BCIP solution (Sigma-Aldrich)
and mixed gently for 2-3 min.
5. Membrane was removed from the solution, washed with distilled water; water was
discarded and the membrane was dried.
Observation and result
After performing the Western blotting procedure a thick band was seen on the nitrocellulose
membrane which corresponds to the β-actin protein which is detected by anti-β-actin
antibody. The molecular weight of β-actin protein is 41 kDa and the position of the band
corresponds to the protein size (figure 4).
Figure 3: Protein on membrane stained with Ponceau S dye
Figure 4: immunodetection of the Protein sample after SDS-PAGE and Western
blotting