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  Section: Biotechnology Methods » Molecular Biology
 
 
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Western Blotting

 
     
 
Content
Molecular Biology
  The Central Dogma
  Protein Synthesis in Cell Free Systems
  Chromosomes
  Polytene Chromosomes of Dipterans
  Salivary Gland Preparation (Squash Technique)
  Extraction of Chromatin
  Chromatin Electrophoresis
  Extraction and Electrophoresis of Histones
  Karyotype Analysis
  In Situ Hybridization
  Culturing Peripheral Blood Lymphocytes
  Microslide Preparation of Metaphases for In-Situ Hybridization
  Staining Chromosomes (G-Banding)
  Nucleic Acids
  Extraction of DNA from Bovine Spleen
  Purification of DNA
  Characterization of DNA
  DNA-Dische Diphenylamine Determination
  Melting Point Determination
  CsCl-Density Separation of DNA
  Phenol Extraction of rRNA (Rat liver)
  Spectrophotometric Analysis of rRNA
  Determination of Amount of RNA by the Orcinol Method
  Sucrose Density Fractionation
  Nucleotide Composition of RNA
  Isolation of Genomic DNA—DNA Extraction Procedure
  Isolation of Genomic DNA from Bacterial Cells
  Preparation of Genomic DNA from Bacteria
  Extraction of Genomic DNA from Plant Source
  Extraction of DNA from Goat Liver
  Isolation of Cotton Genomic DNA from Leaf Tissue
  Arabidopsis Thaliana DNA Isolation
  Plant DNA Extraction
  Phenol/Chloroform Extraction of DNA
  Ethanol Precipitation of DNA
  Isolation of Mitochondrial DNA
  Isolation of Chloroplast DNA
  DNA Extraction of Rhizobium (CsCl Method)
  Isolation of Plasmids
  RNA Isolation
  Preparation of Vanadyl-Ribonucleoside Complexes that Inhibit Ribonuclease Activity
  RNA Extraction Method for Cotton
  Isolation of RNA from Bacteroids
  Isolation of RNA from Free-Living Rhizobia
  Estimation of DNA purity and Quantification
  Fungal DNA Isolation
  Methylene Blue DNA Staining
  Transformation
  Blotting Techniques—Southern, Northern, Western Blotting
  Preparing the Probe
  Southern Blotting (First Method)
  Southern Blotting (Second Method)
  Western Blotting
  Western Blot Analysis of Epitoped-tagged Proteins using the Chemifluorescent Detection Method for Alkaline Phosphatase-conjugated Antibodies
  Southern Blot
  Southern Analysis of Mouse Toe/Tail DNA
  Northern Blotting
  Restriction Digestion Methods—Restriction Enzyme Digests
  Restriction Digestion of Plasmid, Cosmid, and Phage DNAs
  Manual Method of Restriction Digestion of Human DNA
  Preparation of High-Molecular-Weight Human DNA Restriction Fragments in Agarose Plugs
  Restriction Enzyme Digestion of DNA
  Electroelution of DNA Fragments from Agarose into Dialysis Tubing
  Isolation of Restriction Fragments from Agarose Gels by Collection onto DEAE Cellulose
  Ligation of Insert DNA to Vector DNA
  PCR Methods (Polymerase Chain Reaction)
  Polymerase Chain Reaction
  DNA Amplification by the PCR Method

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.

 
     
 
 
     




     
 
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