Western Blotting

Aim
To separate the proteins through SDS-PAGE and detection followed by characterization of proteins through Western blotting.

Introduction
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.

Principle
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 explained below.

SDS-PAGE
Stage 1. Prepare a PAGE gel slab and fix to a vertical electrophoretic apparatus. Treat the sample with suitable buffer and load onto the gel slots.

Stage 2. 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.

Stage 3. 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.


Western Blotting
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 membrane. 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.

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 membrane.


Applications
To characterize proteins and to identify specific antigens for antibodies. Preparation of Reagents

1. Blotting Buffer. Add 25 mL of blotting buffer component A and 25 mL of component B to 150 mL of distilled water.
2. Other Buffers. Dilute the required amount of buffer concentrate to 1X concentration with water.
3. Antibody. Dilute primary Ab and label secondary HRP conjugate in an assay buffer.
4. Substrate. Dilute TMB/H2O2, 10X concentration 10 times with heat just before use.

Procedure

1. Run SDS:Polyacrylamide gel electrophoresis.
2. Electroblot.
3. Assemble the blotting sandwich within the blotting cassette. Care should be taken to avoid air bubbles between the gel and NC membrane.
4. Insert the cassette into the apparatus filled with blotting buffer so that the gel faces the cathode.
5. Connect the power supply and use a voltage of 50 V for 5 hours for blotting.

Immunodetection

1. 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.
2. Suspend the primary antibody in 10 mL with the assay buffer, using a suitable tube.
3. Immerse the blot in the 10 Ab solute and gently agitate for 30 minutes.
4. Wash the blot by immersing it in wash buffer for 3–5 minutes. Repeat 2 more times.
5. Prepare 1:1000 dilutions of labeled 20 Ab in the assay buffer. Prepare sufficient (10 mL) volume of diluted Ab to cover the blot.
6. Immerse the blot in 20 Ab solute and agitate gently for 30 minutes.
7. Wash the blot in wash buffer for 3–5 minutes and repeat 4 times.
8. Immerse the washed blot in 10 mL of substrate solution with gentle shaking. Bands will develop sufficient color within 5–10 min.
9. Remove the blot and wash with distilled water. Dry.
10. 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.