9th&10th lecture Blotting Techniques . Introduction 1) Blotting is a powerful and sensitive technique for identifying the presence of specific biomolecules within a sample. 2) The target molecule that is being sought differentiates subtypes of blotting. 3) The first of these techniques developed was the Southern blot, which is specific for DNA. 4) The second technique is northern blot, which is specific for RNA. 5) The third technique is western blot, which is specific for protein. 6) The fourth technique is eastern blot, which is specific for ……..? 7) The Southern blot, named for Dr. Edwin Southern, who developed it to detect specific DNA sequences in 1975. 8) Subsequently, the method was modified to detect other targets. The nomenclature of these techniques was built around Dr. Sothern's name. 1 Mohanad J.K. Al-Dawah 9th&10th lecture Blotting Techniques . The principles of blotting techniques 1) The basic principles underlying these techniques are virtually identical. 2) First, the target molecules (DNA, RNA, or protein) are separated by a combination of their size and charge using the appropriate method of gel electrophoresis. 3) Second, separated molecules are transferred to a membrane. 4) Third, the membrane is queried with a probe directed against the specific molecule of interest (Figure 1). The principle steps in details First step 1) In the first step, separation of target molecules by gel electrophoresis, large target molecules may first be modified to facilitate movement through the gel. 2) Very large sequences of DNA are processed with restriction endonucleases to cut them into smaller pieces. 3) RNAs often require no additional manipulation, and proteins may be denatured with specific detergents by SDS. 4) The sample is then loaded onto a gel where, in response to application of an electrical charge, molecules vertically separate based on size and charge. Second step 1) In the second step, the molecules are horizontally transferred to a membrane. 2) This done by using an electrical gradient, which maintains the vertical separation established by gel electrophoresis. 3) This step is similar to making a carbon copy, except the molecules, themselves are transferred, rather than an image. 2 Mohanad J.K. Al-Dawah 9th&10th lecture Blotting Techniques Third step 1) Finally, the membrane is treated with probes that bind only the specific molecule being sought (Figure 1); the type of probe varies based upon the target molecule. 2) For nucleic acids (DNA and RNA), the probe is a labeled complementary (antisense) strand that hybridizes to the target sequence. 3) For proteins, a monoclonal antibody (the primary antibody) binds to the protein of interest; this is followed by a second antibody (the secondary antibody), which recognizes the Fc portion of the primary antibody and is itself labeled, allowing specific detection. Notes 1) This “two-step” method is used for cost control because labeling each specific monoclonal antibody would be quite expensive. 2) Because of the use of antibodies, the western blot technique is sometimes referred to as immunoblotting. 3) Regardless of which form of probe is used, the label may be a radioactive isotope or a fluorescent or chromogenic dye, all of which allow detection of the presence of the probe under specific conditions. Figure 1: Overview of blotting steps 3 Mohanad J.K. Al-Dawah 9th&10th lecture Blotting Techniques . Southern blotting 1) Southern blot named after Sir Edwin Southern developed in 1975 2) This method involves separation, transfer and hybridization. 3) The Southern blot is used to detect the presence of a particular piece of DNA in a sample. 4) The DNA detected can be a single gene, or it can be part of a larger piece of DNA such as a viral genome. 5) The key to this method is Hybridization. 6) Hybridization is the process of forming a double-stranded DNA molecule between a single-stranded DNA probe and a single- stranded target patient DNA. Steps of southern blotting 1) The DNA to be analyzed, such as the total DNA of an organism, is digested to completion with a restriction enzyme. 2) The complex mixture of fragments is subjected to gel electrophoresis to separate the fragments according to size. 3) The restriction fragments present in the gel are denatured with alkali and transferred onto a nitrocellulose filter or nylon membrane by blotting. 4) This step preserves the distribution of the fragments in the gel, creating a replica of the gel on the filter 5) The filter is incubated under hybridization conditions with a specific radiolabeled DNA probe. 6) The probe hybridizes to the complementary DNA restriction fragment. 7) Excess probe is washed away and the probe bound to the filter is detected by autoradiography, which reveals the DNA fragment to which the probe hybridized. 4 Mohanad J.K. Al-Dawah 9th&10th lecture Blotting Techniques Advantages of southern blotting 1) Effective way to detect a specific DNA sequence in a large, complex sample of DNA. 2) Can have results that are just distinctive individual specific as real human fingerprints. 3) More suitable than various other tests to analyze long stretches of DNA. 4) Can be used to quantify the amount of DNA present. 5) Used as definitive test when identify genetically modified organisms. 6) Cheaper than DNA sequencing. Disadvantages of southern blotting 1) More expensive than most other tests. 2) Complex, labor intensive and time consuming. Northern blotting technique 1) Northern blotting is a technique for detection of specific RNA sequences. 2) Northern blotting was developed in 1977 by James Alwine, David Kemp and George Stark at Stanford University and was named such by analogy to Southern blotting. Steps of northern blotting technique 1) RNA is isolated from several biological samples RNA is more susceptible to degradation than DNA. 2) Samples are loaded on gel and the RNA samples are separated according to their size on an agarose gel. 3) The gel is then blotted on a nylon membrane or a nitrocellulose filter paper by creating the sandwich arrangement. 5 Mohanad J.K. Al-Dawah 9th&10th lecture Blotting Techniques 4) The membrane is placed in a dish containing hybridization buffer with a labeled probe. 5) The membrane is washed to remove unbound probe. 6) The labeled probe is detected via autoradiography or via a chemiluminescence reaction. Advantages of northern blotting 1) It is a widely accepted and well-regarded method, often a gold standard 2) Northern blotting is a straightforward method. 3) Often it is used as a confirmation or check. 4) It is a versatile protocol as it can allow the usage of many types of probes (vs Real time PCR) including radiolabeled and non- radiolabeled, in vitro transcribed RNA and even oligonucleotides such as primers. Disadvantages of northern blotting 1) Often radioactivity is used. This prevents ease of performing it, use and disposal. 2) Usually takes a long time from sample preparation through to detection. 3) The standard northern blot method is relatively less sensitive than nuclease protection assays and RT-PCR. Western blotting technique 1) Western blotting 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. 2) This technique introduced by Towbin, et al. in 1979. 6 Mohanad J.K. Al-Dawah 9th&10th lecture Blotting Techniques 3) In Western blotting, the molecule of interest is a protein and the probe is typically an antibody raised against that particular protein. 4) The SDS PAGE technique is a prerequisite for Western blotting. Steps of western blotting 1) A protein sample is subjected to electrophoresis on an SDS- polyacrylamide gel. 2) Electro-blotting transfers the separated proteins from the gel to the surface of a nitrocellulose membrane. 3) The blot is incubated with a generic protein, which binds to any remaining sticky places on the nitrocellulose. 4) An antibody that is specific for the protein of interest (the primary antibody - Ab1) is added to the nitrocellulose sheet and reacts with the antigen. 5) Only the band containing the protein of interest binds the antibody, forming a layer of antibody molecules. 6) Following several rinses for removal of non-specifically bound Ab1, the Ab1-antigen complex on the nitrocellulose sheet is incubated with a second antibody (Ab2), which specifically recognizes the Fc domain of the primary antibody and binds it. 7) Ab2 is radioactively labeled, or is covalently linked to a reporter enzyme, which allows visualizing the protein-Ab1-Ab2 complex. Advantages of western blotting 1) It provides more specific results than the ELISA protocol by providing additional information regarding protein size and multiple bands. 2) Western blot test is referred to as the 'Gold Standard', meaning it trumps any other positive tests. 7 Mohanad J.K. Al-Dawah 9th&10th lecture Blotting Techniques 3) Western blotting tells you how much protein has accumulated in cells. Disadvantages of western blotting 1) It is time-consuming (compared to ELISA). 2) If a protein is degraded quickly, Western blotting will not detect it well. 3) It might also be more costly. New techniques compared with blotting Name Target Probes used Newer alternative techniques Southern DNA Complementary (antisense) PCR, real-time PCR, FISH sequence of DNA or RNA Northern RNA Complementary sequence of Real-time PCR DNA or RNA Western Protein Monoclonal antibody ELISA, immunohistochemistry, immunofluorescence, flow cytometry . Comparison between blotting techniques Characters Southern Northern Western Molecule detected DNA RNA Protein Gel electrophoresis Agarose gel Formaldehyde Polyacrylamide gel agarose gel Blotting method Capillary transfer Capillary transfer Electric transfer Probes Radioactive or Radioactive or primary antibody nonradioactive nonradioactive Detection system Autoradiography Autoradiography Chemiluminescent Chemiluminescent Chemiluminescent Colorimetric Colorimetric Colorimetric 8 Mohanad J.K.