To separate the proteins through SDS-PAGE and detection followed by
characterization of proteins through Western blotting.
Western blotting (also known as protein- or immunoblotting) is a rapid and
sensitive assay for detection and characterization of proteins. It works by
exploiting the specificity inherent in Ag-Ab recognition. It is used to identify
specific antigens recognized by polyclonal or monoclonal antibodies. Western
blotting is carried out along with protein (antigen) separation in gel by
electrophoresis and the blot development.
It is essentially a combination of 3 techniques:
- Electrophoresis (PAGE)
- Western blotting
- Immunochemical detection.
Identification of protein separated by gel electrophoresis is limited by the small
pore size of the gel, as the macromolecule probe for protein analysis cannot
permeate the gel. This limitation is overcome by blotting the protein into an
adsorbent porous membrane. The apparatus consists of a tank containing buffer,
in which is located a cassette. Clamping the gel and the membrane tightly
together, a current is applied from electrodes, and repeated on either side of the
cassette to avoid heating effects. The proteins are separated according to their
electrophoresis mobility and blotted onto the membrane identified, using suitable
immunochemicals to locate the protein of interest. The individual techniques are
Prepare a PAGE gel slab and fix to a vertical electrophoretic apparatus.
Treat the sample with suitable buffer and load onto the gel slots.
Apply electric current. After a few minutes, proteins in the sample
migrate according to their electrophoretic mobility in the stacking gel. The
stacking gel has a polyacrylamide concentration resulting in higher pore size
and a lower pH of 7. This enables the protein to concentrate into sharp bands
due to isotachopharesis, or band-sharpening effect. At the end of the stacking
gel, it meets the separating gel, which has a higher polyacrylamide concentration
and higher pH. In the separating gel, the proteins travel according to their size.
When the dye front reaches the bottom of the separating gel, the
proteins in the sample are resolved depending on their size. However the
protein cannot be visualized directly. The gel needs to be stained with suitable
stainer to visualize all the proteins. The identification of protein of interest can
be done using a suitable probe and a developing system.
Blotting is the transfer of resolved proteins from the gel to surface of suitable
membrane. This is done commonly by electrophoresis (known as electroblotting).
In this method, the transfer buffer has a low ionic strength which allows
electrotransfer of proteins. Methane in the buffer increases binding of proteins
to niotrocellulose and reduces gel smelling during transfer. The use of the
membrane as a support for protein enables the case of manipulation efficient
washing and faster reactions during the immunodetection, as proteins are more
accessible for reaction.
(a) The membrane is in close contact with PAGE gel containing proteins. The
proteins are electrotransferred to nitrocellulose membrane.
(b) At the end of electrotransfer, all proteins would have migrated to the NC
The protein was transferred to the corresponding position on the membrane
as on the gel. A mirror image of the gel was formed. However, the protein
location and detection can only be assessed after immunodetection.
The transferred proteins are bound to the surface of NC membrane and are
accessible for reaction with immunochemical reagents. All the unoccupied sites
on the membrane are blocked with inert proteins, detergents, or other suitable
blocking agents. The membrane is then probed with a primary antibody and a
suitable substrate so the enzyme identifies the ag-ab complex form on the
To characterize proteins and to identify specific antigens for antibodies.
Preparation of Reagents
- Blotting Buffer. Add 25 mL of blotting buffer component A and 25 mL of
component B to 150 mL of distilled water.
- Other Buffers. Dilute the required amount of buffer concentrate to 1X
concentration with water.
- Antibody. Dilute primary Ab and label secondary HRP conjugate in an
- Substrate. Dilute TMB/H2O2, 10X concentration 10 times with heat just
- Run SDS:Polyacrylamide gel electrophoresis.
- Assemble the blotting sandwich within the blotting cassette. Care should
be taken to avoid air bubbles between the gel and NC membrane.
- Insert the cassette into the apparatus filled with blotting buffer so that the
gel faces the cathode.
- Connect the power supply and use a voltage of 50 V for 5 hours for
- Remove the NC membrane gently from the cassette and place it in the
blocking buffer for 2 hours at room temperature, or overnight in the cold.
- Suspend the primary antibody in 10 mL with the assay buffer, using a
- Immerse the blot in the 10 Ab solute and gently agitate for 30 minutes.
- Wash the blot by immersing it in wash buffer for 3–5 minutes. Repeat 2
- Prepare 1:1000 dilutions of labeled 20 Ab in the assay buffer. Prepare
sufficient (10 mL) volume of diluted Ab to cover the blot.
- Immerse the blot in 20 Ab solute and agitate gently for 30 minutes.
- Wash the blot in wash buffer for 3–5 minutes and repeat 4 times.
- Immerse the washed blot in 10 mL of substrate solution with gentle shaking.
Bands will develop sufficient color within 5–10 min.
- Remove the blot and wash with distilled water. Dry.
- Although the colored hands fade with time, the rate of color loss can be
retarded if the blots are kept in the dark.
Interpretation and Result
The proteins separated through the SDS-PAGE have been successfully transferred
onto the nitrocellulose membrane and the transferred proteins detected by
immunodetection, which was confirmed by the development of color bands on
the nitrocellulose membrane.