7 DNA Microarray & Western Blotting

• In DNA microarray technology, the filters (membranes) have been replaced by a microscopic slide chemically treated

  • e.g., nitrocellulose coated microscopic glass slides
• Macroarrays use a nylon or nitrocellulose membrane matrix on which nucleic acid probes have been deposited.

  • Samples are radioactively labeled and hybridized in parallel.
  • Detection is best accomplished through phosphorimaging (although film autoradiography can be employed).
• Microarrays use a silicon or plastic matrix and a fluorescence-based labelling scheme in which both control and experimental samples are hybridized to the same array in a competitive manner.

  • Fluorescent scanners are used for detection.
• In Microarrays of pre-synthesized nucleic acids, individual DNA clones or oligonucleotides are spotted at individual locations using a robot
• In Microarrays of oligonucleotides synthesized In Situ, DNA chips are constructed by taking advantage of photolithography and the chemistry of oligonucleotide synthesis.

  • Digital imaging software is used to analyze a signal emitted by each spot on the microarray.
• DNA microarray technology has very important applications in biomedical research and diagnostic approaches.
• Western blot is an immunoblotting technique which rely on the specificity of binding between a molecule of interest and a probe to allow detection of the molecule of interest in a mixture of many other similar molecules
• The SDS-PAGE technique is a prerequisite for Western blotting
• Proteins from all sources can be used for western blotting such as from a single cell, whole tissues, extracellular matrices, and biological fluids
• Polyacrylamide gel electrophoresis is mainly used for the separation of protein particles.
• Sodium dodecyl sulfate is added to the gel and running buffer to denature the protein molecules which will ensure that your proteins are separated solely on the basis of size and not on three-dimensional structure
• Molecular weight markers are used to define the size of proteins run in a gel; composed of different proteins of known size.
• As proteins are not directly visible in the gel, the gel must be stained.
• Proteins are transferred from the gel to a solid support membrane such as nitrocellulose or polyvinylidene difluoride
• The proteins transferred from the gels are immobilized, which makes it possible to detect the proteins on the membrane using specific antibodies
• Electro-blotting is mainly used which uses electric field to pull proteins from the gel onto the PVDF or nitrocellulose membrane
• Two main classes of blocking agents are commonly used for western blotting → proteins and non-ionic detergents
• Western blotting protocols usually uses a non-labeled primary antibody directed against the target protein and labeled secondary antibody directed against the constant region of the primary antibody
• Secondary antibody in antibody probing serves not only as a carrier of a label but also as a mechanism to amplify the emitted signals.

  • They are polyclonal so they can bind primary antibodies and different epitopes simultaneously.
• Alkaline phosphatase and horseradish peroxidase are the two most used enzymes for protein detection using chromogenic substrates.
• Enzymatic detection in western blotting requires the addition of a substrate that emits light when it reacts with an enzyme conjugated to a secondary antibody.
• Fluorescence-based detection in western blotting requires the excitation of fluorophore using a light source of a specific wavelength causing light emission.
• Detection of signals, using either Xray film, scanners or a charge coupled device camera-based imager, results in image containing visible protein bands.

  • The presence and the amount of a protein of interest is estimated by visual analysis, and the size can be determined by comparison to a known molecular weight marker.
• Western blot

  • most sensitive and specific test for determining size and amount of protein present in any material
  • confirmatory test for HIV
  • definitive test for Creutzfeldt-Jakob Disease, Lyme disease, Hepatitis B infection and HSV-2 (Herpes Type 2) infection.