Western blotting or Immunoblotting, is a well established and widely used technique for the detection and analysis of proteins. The method is based on building an antibody:protein complex via specific binding of antibodies to proteins immobilized on a membrane and detecting the bound antibody with one of several detection methods. The Western blotting method was first described in 1979 and has since become one of the most commonly used methods in life science research.
For Western Blotting Protocol click here
Types of Membranes Used in Western Blotting
Nylon membranes have high mechanical strength compared to the nitrocellulose membranes. The mechanism of protein binding to the membrane is by electrostatic interaction. The protein binding capacity of nylon membrane is in the range of 150 ug / cm2 to 200 ug /cm2.
Types of Membranes Used in Western Blotting
There are three types of membranes widely used in western blotting these are
- Nitrocellulose Membrane
- Nylon Membrane
- Polyvinylidene fluoride (PVDF) Membrane
Nitrocellulose Membrane
Membrane Pore Size Available: 0.45µm , 0.22 µm, 0.1 µm.
Nitrocellulose Membrane having smaller pore size 0.22 µm and 0.1µm are recommended for immunoblotting of smaller proteins(<14 div="" kda="">
Nitocellulose membranes have a protein Binding and retaining capacity of 80 µg/ cm^2 to 250µg/ cm^2.
Mechanism of Protein Binding to Nitrocellulose Membrane
Protein binds to the Nitrocellulose membrane by means of Hydrophobic Interaction, there is also hydrogen bond formation between the nitro group of the nitrocellulose membrane and the amino acid side chains of the protein.
Salts present in the western blot transfer buffer and the methanol used for wetting the membrane causes the partial dehydration of the proteins which in-turn results in the long lasting bonding between the membrane and the protein.
Advantages of Nitrocellulose Membrane
- The main advantage of using Nitrocellulose Membrane is that which gives very low background.
Disadvantages of Nitrocellulose Membrane
- Low Mechanical Strength – Becomes brittle and difficult to handle when dry.
- Cannot be used for multiple stripping and reprobing.
Nylon Membrane
Nylon membranes have high mechanical strength compared to the nitrocellulose membranes. The mechanism of protein binding to the membrane is by electrostatic interaction. The protein binding capacity of nylon membrane is in the range of 150 ug / cm2 to 200 ug /cm2.
Advantages of Nylon Membranes
- Multiple probing of antibodies possible using Nylon membranes
Disadvantages of Nylon Membranes
- Sensitive technique is not available for staining protein bound to the membrane.
- Since free sites cannot blocked completely, Non-specific binding antibodies to the membrane occurs resulting in high background.
- Extended blocking steps may be required in order to achieve satisfactory results.
Polyvinylidene fluoride (PVDF) Membrane
PVDF membranes are mechanically strong. The mechanism of protein binding to the membrane is by hydrophobic interaction. Wetting the PVDF membrane with methanol prior to transfer shown to have increase the protein binding. The protein binding capacity of these membranes are in the range of 170 ug/cm2 to 200 ug/cm2. Proteins binds more tightly to the PVDF membrane than to the Nitrocellulose membranes. The tight binding helps in retaining the protein during the washing steps.
Advantages of PVDF Membranes
- High mechanical Strength
- Staining is easy.
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