Blotting Techniques

Blotting techniques

Southern Blotting

A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.

The method is named after its inventor, the British biologist Edwin Southern. Other blotting methods (i.e., western blot, northern blot, eastern blot, southwestern blot) that employ similar principles, but using RNA or protein, have later been named in reference to Edwin Southern’s name.

Method

  1. Restriction endonucleases are used to cut high-molecular-weight DNA strands into smaller fragments.
  2. The DNA fragments are then electrophoresed on an agarose gel to separate them by size.
  3. If some of the DNA fragments are larger than 15 kb, then prior to blotting, the gel may be treated with an acid, such as dilute HCl. This depurinates the DNA fragments, breaking the DNA into smaller pieces, thus allowing more efficient transfer from the gel to membrane.
  4. If alkaline transfer methods are used, the DNA gel is placed into an alkaline solution (typically containing sodium hydroxide) to denature the double-stranded DNA. The denaturation in an alkaline environment may improve binding of the negatively charged thymine residues of DNA to a positively charged amino groups of membrane, separating it into single DNA strands for later hybridization to the probe and destroys any residual RNA that may still be present in the DNA.
  5. A sheet of nitrocellulose (or, alternatively, nylon) membrane is placed on top of the gel. Pressure is applied evenly to the gel to ensure good and even contact between gel and membrane. Buffer transfer by capillary action from a region of high water potential to a region of low water potential (usually filter paper and paper tissues) is then used to move the DNA from the gel on to the membrane’
  6. The membrane is then baked in a vacuum or regular oven at 80 °C for 2 hours or exposed to ultraviolet radiation to permanently attach the transferred DNA to the membrane.
  7. The membrane is then exposed to a hybridization probe—a single DNA fragment with a specific sequence whose presence in the target DNA is to be determined. The probe DNA is labelled so that it can be detected, usually by incorporating radioactivity or tagging the molecule with a fluorescent or chromogenic dye. In some cases, the hybridization probe may be made from RNA, rather than DNA.
  8. After hybridization, excess probe is washed from the membrane and the pattern of hybridization is visualized on X-ray film by autoradiography in the case of a radioactive or fluorescent probe, or by development of color on the membrane if a chromogenic detection method is used.SOUTHERN BLOTT

Applications

Southern transfer may be used for homology-based cloning on the basis of amino acid sequence of the protein product of the target gene. Oligonucleotides are designed that are similar to the target sequence. The oligonucleotides are chemically synthesised, radiolabeled, and used to screen a DNA library, or other collections of cloned DNA fragments. Sequences that hybridise with the hybridisation probe are further analysed, for example, to obtain the full length sequence of the targeted gene.

Southern blotting can also be used to identify methylated sites in particular genes. Particularly useful are the restriction nucleases MspI and HpaII, both of which recognize and cleave within the same sequence. However, HpaII requires that a C within that site be methylated, whereas MspI cleaves only DNA unmethylated at that site.

Northern Blotting

The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample.

Northern blotting involves the use of electrophoresis to separate RNA samples by size and detection with a hybridization probe complementary to the entire target sequence. The term ‘northern blot’ actually refers specifically to the capillary transfer of RNA from the electrophoresis gel to the blotting membrane. However, the entire process is commonly referred to as northern blotting. The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University. Northern blotting takes its name from its similarity to the first blotting technique, the Southern blot, named for biologist Edwin Southern. The major difference is that RNA, rather than DNA, is analyzed in the northern blot.

Procedure

A general blotting procedure starts with extraction of total RNA from a homogenized tissue sample or from cells. RNA samples are then separated by gel electrophoresis. Since the gels are fragile and the probes are unable to enter the matrix, the RNA samples, now separated by size, are transferred to a nylon membrane through a capillary or vacuum blotting system.

A nylon membrane with a positive charge is the most effective for use in northern blotting since the negatively charged nucleic acids have a high affinity for them. Once the RNA has been transferred to the membrane, it is immobilized through covalent linkage to the membrane by UV light or heat. After a probe has been labeled, it is hybridized to the RNA on the membrane. Experimental conditions that can affect the efficiency and specificity of hybridization include ionic strength, viscosity, duplex length, mismatched base pairs, and base composition. The membrane is washed to ensure that the probe has bound specifically and to avoid background signals from arising. The hybrid signals are then detected by X-ray film and can be quantified by densitometry.

Northern_blot6

Applications

Northern blotting is used to observe a particular gene’s expression pattern between tissues, organs, developmental stages, environmental stress levels, pathogen infection, and over the course of treatment.

The technique has been used to show overexpression of oncogenes and downregulation of tumor-suppressor genes in cancerous cells when compared to ‘normal’ tissue, as well as the gene expression in the rejection of transplanted organs.

It includes the detection of RNA size, the observation of alternate splice products, the use of probes with partial homology, the quality and quantity of RNA.

 

 

 

Western Blotting

The western blot called the protein immunoblot is a widely used analytical technique used to detect specific proteins in a sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide. The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are stained with antibodies specific to the target protein. The gel electrophoresis step is included in Western blot analysis to resolve the issue of the cross-reactivity of antibodies.

This method is used in the fields of molecular biology, immunogenetics and other molecular biology disciplines. A number of search engines, such as CiteAb, are available that can help researchers find suitable antibodies for use in Western Blotting.

Other related techniques include dot blot analysis, immunohistochemistry and immunocytochemistry where antibodies are used to detect proteins in tissues and cells by immunostaining, and enzyme-linked immunosorbent assay (ELISA).

Steps

Tissue preparation

Samples can be taken from whole tissue or from cell culture. Solid tissues are first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), or by sonication. Cells may also be broken open by one of the above mechanical methods. However, virus or environmental samples can be the source of protein

Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins. Protease and phosphatase inhibitors are often added to prevent the digestion of the sample by its own enzymes. Tissue preparation is often done at cold temperatures to avoid protein denaturing and degradation.

A combination of biochemical and mechanical techniques – comprising various types of filtration and centrifugation – can be used to separate different cell compartments and organelles.

Gel electrophoresis

The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point (pI), molecular weight, electric charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel. This is a very useful way to identify a protein.

SDS-PAGE (SDS polyacrylamide gel electrophoresis) maintains polypeptides in a denatured state once they have been treated with strong reducing agents to remove secondary and tertiary structure and thus allows separation of proteins by their molecular weight. Sampled proteins become covered in the negatively charged SDS and move to the positively charged electrode through the acrylamide mesh of the gel.

Samples are loaded into wells in the gel. One lane is usually reserved for a marker or ladder, a commercially available mixture of proteins having defined molecular weights, typically stained so as to form visible, coloured bands. When voltage is applied along the gel, proteins migrate through it at different speeds dependent on their size.

Transfer

In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). The primary method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane.

As a result of either “blotting” process, the proteins are exposed on a thin surface layer for detection. Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein.

The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dyes. Ponceau S is the more common of the two, due to its higher sensitivity and water solubility.

Blocking

Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein – typically 3-5% Bovine serum albumin (BSA) or non-fat dry milk (both are inexpensive) in Tris-Buffered Saline (TBS). The protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached.

Detection

During the detection process the membrane is “probed” for the protein of interest with a modified antibody which is linked to a reporter enzyme; when exposed to an appropriate substrate, this enzyme drives a colourimetric reaction and produces a color.

Primary antibody

After blocking, a dilute solution of primary antibody (generally between 0.5 and 5 micrograms/mL) is incubated with the membrane under gentle agitation. Typically, the solution is composed of buffered saline solution with a small percentage of detergent, and sometimes with powdered milk or BSA. The antibody solution and the membrane can be sealed and incubated together for anywhere from 30 minutes to overnight. It can also be incubated at different temperatures, with higher temperatures being associated with more binding, both specific and non-specific.

Secondary antibody

After rinsing the membrane to remove unbound primary antibody, the membrane is exposed to another antibody, directed at a species-specific portion of the primary antibody. The secondary antibody is usually linked to biotin or to a reporter enzyme such as alkaline phosphatase or horseradish peroxidase. This means that several secondary antibodies will bind to one primary antibody and enhance the signal.

Most commonly, a horseradish peroxidase-linked secondary is used to cleave a chemiluminescent agent, and the reaction product produces luminescence in proportion to the amount of protein. A sensitive sheet of photographic film is placed against the membrane, and exposure to the light from the reaction creates an image of the antibodies bound to the blot.

The enzyme can be provided with a substrate molecule that will be converted by the enzyme to a colored reaction product that will be visible on the membrane.

WesternBlotFinal

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