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Blotting Techniques

Southern blotting

A Southern blot is a method  used  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.


The procedure

High molecular weight DNA strands are cut into smaller fragments by restriction endonucleases. The DNA fragments are separated by size by agarose gel electrophoresis and then transferred to a nitrocellulose membrane which is placed on the top of the gel (Figure-1). In Sourthen blotting, before transfer, DNA is usually denatured with alkali for denaturation of the double stranded DNA.  The denaturation in an alkaline environment may improve binding of the negatively charged DNA to a positively charged 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.  After transfer of the DNA fragments to the nitrocellulose membrane which is done by capillary action or may be by electrotransfer, vacuum transfer or centrifugation, the membrane is then baked in a vacuum or regular oven at 80 °C for 2 hours to permanently attach the transferred DNA to the membrane. 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. 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.

 

Figure 1: Southern blotting
Figure 1: Southern blotting

      

       The hybridization and washing conditions are critical. If the probe and target are 100% identical in sequence, then a high stringency hybridization can be carried out. The stringency is determined by the hybridization temperature and the salt concentration in the hybridization buffer. For probes that don't match the target completely, the stringency must be reduced to a level that allows imperfect hybrids to form. If the stringency of the hybridization is too low, then the probe may bind to too many sequences to be useful. Formamide can be included in the hybridization buffer to reduce the actual hybridization temperature by about 25°C, from the usual 68°C to the more convenient 43°C.


Applications

Southern hybridization can also be used to locate the exact position of a cloned gene within a recombinant DNA molecule. This is important as often the cloned DNA fragment is relatively large (40 kb for a cosmid vector) whereas the gene of interest, contained somewhere in the cloned fragment, may be less than 1kb in size. Southern blots of cloned genomic DNA fragments can be probed with cDNA molecules to find which parts of the genomic clone correspond to the cDNA fragment. If the Southern blot contains genomic DNA fragments from the whole genome, the probe will give information about the size of the fragment the gene is on the genome and how many copies of the gene are present in the genome.

 

Northern blotting

Northern blotting, the name was extrapolated from Southern blotting. The northern blot is a technique used  to study gene expression by detection of RNA (or isolated mRNA) in a sample.

 

The Procedure

The nucleic acid molecules (RNA samples) are separated by agarose gel electrophoresis and then transferred to a nitrocellulose membrane but for RNA in Northern blotting, alkali denaturation is not necessary and would in any case hydrolyze the molecules. 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. The transfer buffer used for the blotting usually contains formamide because it lowers the annealing temperature of the probe-RNA interaction, thus preventing RNA degradation by high temperatures. Once the RNA has been transferred to the membrane, it is immobilized through covalent linkage to the membrane by UV light or heat (Figure-2). 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.  

 

Figure 2: Outline of the general procedure for RNA detection by northern blotting
Figure 2: Outline of the general procedure for RNA detection by northern blotting


Applications

Northern blots give information about the size of the mRNA and any precursors, and can be useful to determine whether a cDNA clone used as a probe is full-length or whether it is one of a family of related transcripts. Northern blots can help to identify whether a genomic clone has regions that are transcribed and, if the RNA on the blot is made from different tissues, where these transcripts are made. With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression levels during differentiation, morphogenesis, as well as abnormal or diseased conditions.

 

Western blotting

Identification of a specific protein in a complex mixture of proteins can be done by a technique known as western blotting. Western blotting (also called immunoblotting because an antibody is used to specifically detect its antigen) was introduced by Towbin, et al. in 1979 and is now a routine technique for protein analysis. The procedure for Western blotting is given below.

 

Gel electrophoresis

 In Western blotting, first a protein mixture is separated by electrophoresis on an SDS-polyacrylamide gel (SDS-PAGE), a slab gel infused with sodeum dodecyl sulphate (SDS), a dissociating agent (Figure-3). Proteins are commonly separated using polyacrylamide gel electrophoresis (PAGE) to characterize individual proteins in a complex sample or to examine multiple proteins within a single sample. When combined with Western blotting, PAGE is a powerful analytical tool providing information on the mass, charge, purity or presence of a protein. Several forms of PAGE exist and can provide different types of information about the protein(s). 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 (e.g. disulfide bonds [S-S] to sulfhydryl groups [SH and SH]) 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. Smaller proteins migrate faster through this mesh and the proteins are thus separated according to size (usually measured in kilodaltons, kDa). The concentration of acrylamide determines the resolution of the gel - the greater the acrylamide concentration the better the resolution of lower molecular weight proteins. The lower the acrylamide concentration the better the resolution of higher molecular weight proteins. Proteins travel only in one dimension along the gel for most blots. Samples are loaded into wells in the gel. When voltage is applied along the gel, proteins migrate into it at different speeds. These different rates of advancement (different electrophoretic mobilities) separate into bands within each lane.

 

Transfer of proteins to a membrane

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). There are a variety of methods that have been used for this process, including diffusion transfer, capillary transfer, heat-accelerated convectional transfer, vacuum blotting transfer and electroelution. In capillary transfer, the membrane is placed on top of the gel, and a stack of filter papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it.But this method of transfer is very time consuming.  The transfer method that is most commonly used for proteins is electroelution or electrophoretic transfer because of its speed and transfer efficiency. Electrophoretic transfer of proteins involves placing a protein-containing polyacrylamide gel in direct contact with a piece of nitrocellulose or other suitable, protein-binding support and "sandwiching" this between two electrodes submerged in a conducting solution. When an electric field is applied, the proteins move out of the polyacrylamide gel and onto the surface of the membrane, where the proteins become tightly attached (Figure-3). The result is a membrane with a copy of the protein pattern that was originally in the polyacrylamide gel. 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.

 

Blocking non-specific sites

After the transfer of the proteins from the gel, the remaining surface of the membrane is blocked to prevent non-specific binding of the detection antibodies during subsequent steps. 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  in Tris-Buffered Saline (TBS), with a minute percentage of detergent such as Tween 20 or Triton X-100. The protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached. Thus, when the antibody is added, there is no room on the membrane for it to attach other than on the binding sites of the specific target protein. This reduces "noise" in the final product of the western blot, leading to clearer results, and eliminates false positives.

 

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 colour.
 

Incubation with the primary antibody

Western blotting is typically performed by probing the blocked membrane with a primary antibody that recognizes a specific protein or epitope on a group of proteins (i.e., SH2 domain or phosphorylated tyrosine). The choice of a primary antibody for a Western blot will depend on the antigen to be detected and what antibodies are available to that antigen.

       After blocking, a dilute solution of primary antibody  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. If incubating in blocking buffer overnight, it is imperative to incubate at 4°C or contamination will incur and thus destruction of the protein (especially phospho groups). Agitation of the antibody is recommended to enable adequate homogenous covering of the membrane and prevent uneven binding.
 

Incubation with 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. A wide variety of labeled secondary detection reagents can be used for Western blot detection. 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.

 

Methods of detection

Enzymatic labels are most commonly used for Western blotting and, although they require extra steps, can be extremely sensitive when optimized with an appropriate substrate. Alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two enzymes used most extensively as labels for protein detection. The high activity rate, good stability, low cost and wide availability of substrates make HRP the enzyme of choice for most applications. After binding of the enzyme-antibody conjugate, addition of a chromogenic substrate (Figure-3) that produces a highly coloured and insoluble product causes the appearance of a coloured band at the site of the target antigen. The site of the protein of interest can be determined with much higher sensitivity if a chemiluminescent compound along with suitable enhancing agents is used to produce light at the antigenic site.

The second method method of secondary antibody detection utilizes a near-infrared (NIR) fluorophore-linked antibody. Light produced from the excitation of a fluorescent dye is static, making fluorescent detection a more precise and accurate measure of the difference in signal produced by labeled antibodies bound to proteins on a western blot. Proteins can be accurately quantified because the signal generated by the different amounts of proteins on the membranes is measured in a static state, as compared to chemiluminescence, in which light is measured in a dynamic state. The use of fluorophore-conjugated antibodies in  immunoassays requires fewer steps because there is no substrate development step in the assay. This method requires special equipment in order to detect and document the fluorescent signal due to the need for an excitation light source.

Another alternative is to use a radioactive label rather than an enzyme coupled to the secondary antibody. If the protein of interest was bound by a radioactive antibody, its position on the blot can be determined by exposing the membrane to a shit of x-ray film, a procedure called autoradiography.

 

 

Figure 3: Method of Western blotting
Figure 3: Method of Western blotting

 

Applications

  • Western blotting can  be used to identify a specific antibody in a mixture. In this case, known antigens of well-defined molecular weight are separated by SDS-PAGE and blotting onto nitrocellulose. The separated bands of known antigens are then probed with the sample suspected of containing antibody specific for one or more of these antigens. Reaction of an antibody with a band is detected by using either radiolabeled or enzyme linked secondary antibody that is specific for the species of the antibodies in the test sample. The most widely used application of this procedure is in confirmatory testing for HIV, where Western blotting is used to determine whether the patient has antibodies that react with one or more viral proteins.
  • A western blot is also used as the definitive test for mad cow disease.
  • Western blot can also be used as a confirmatory test for Hepatitis B infection.
     

 

 

References

1. Brown, T.A. (1998). Gene cloning an introduction, 3rd Edn. Stanley Thornes (Publishers) Ltd.

2. Kuby, Janis etal.(2003). Immunology, 5th Edn. W.H. Freeman & Company Publishers.

3. Turner Phil, McLennan Alexander, Bates Andy and White Mike (2005). Characterization of clones.Instant notes(160-161), Molecular Biology. Taylor& Francis Group

4. www.wikipedia.org
 

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