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Saturday, March 15, 2014

Electrochemical Detection Methods

There are 3 types of Electrochemical Detection Methods
  1. Amperometric Method
  2. Potentiometric Method 
  3. Conductimetric Method
Amperometric Detection Method

Amperometric Method of detection is by measuring the current. In amperometric detection, oxidation or reduction of analyte at the working electrode takes place. The oxidation or reduction of the analyte resulting in the gain or loss of electrons at the electrode surface. The electric current generated due to the electrons passed to or from the electrode can be recorded and it will be proportional to the analyte concentration.

Oxidation is mostly employed mode since dissolved oxygen interferes in the reduction.

Principle of Ampermetric Detection

The analyte bound to the receptor will undergo oxidation reduction reaction that can be quantified by measuring the electron exchange from working electrode to the counter electrode.

The current measured in the amperometric detection is relate to

  • Electrode Area, 
  • No of electrons exchanged, 
  • Faraday constant, 
  • Diffusion coefficient,
  • Thickness of the diffusion layer 
  • Concentration of the species
  • Glucose Biosensors

Potentiometric Detection Method

Potentiometric Detection method is based on the measurement of Nernst potential either across an ion selective barrier or at an electrode surface with respect to a reference electrode.

Potentiometric Detection Measures the voltage between the working electrode and the reference electrode

In potentiometric Detection method, ion selective electrodes are used as sensors.


Oxygen Sensors

pH determination

Conductimetric Detection Method

Conductimetric Detection is based on the measurement of the conductivity
or resistance of a solution between two indicator electrodes 
when a small current is applied to them.

Most widely used in ion chromatography.

Thursday, March 6, 2014

Western Blot Membrane Types and uses

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

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.