immunoblotting techniques.pptx

OTECHNOLOGY IS CHALLENGING SUBJECT TO TEACH AND UNDERSTAND ALSO .....THEIR INTERESTING PART IS TO LEARN ABOUT MICROBIAL GENETICS AND THEIR METHODS OF GENE TRANSFER

1. MICROBIAL GENETICS By TEJASWINI L. ASAWE ASSISTANT PROFESSOR SIDHHIS INSTITUTE OF PHARMACY THANE

2. DEFINITION OF BLOTTING Visualization of specific DNA , RNA & protein among many thousands of contaminating molecules requires the convergence of number of techniques which are collectively termed BLOT transfer .

3. Blotting technique Southern Blot It is used to detect DNA. Northern Blot It is used to detect RNA. Western blot It is used to detect protein Types

4. Blotting Blotting is a method of transferring proteins , DNA or RNA , onto a carrier Ex. Nitrocellulose , nylon membrane The southern blot is used for transferring DNA The Northern Blot is used to detect RNA The Western Blot is used to detect proteins.

5. Immunobloting A laboratory procedure, in which proteins that have been separated by electrophoresis are transferred to a membrane of nitrocellulose or another material and are identified by their reaction with labeled antibodies. Immunoblotting allows detection of a protein antigen immobilized on the protein-retaining membrane support such as nitrocellulose or polyvinylidene fluoride (PVDF). The detection of the protein of interest relies on the binding of an antibody that specifically recognizes the protein of interest exposed on the membrane

6. WESTERN BLOTTING Western blotting is widely used analytical technique in molecular biology to detect specific protein in a sample of tissue extract . It works on the principle of gel electrophoresis. Proteins are separated based on their size on polyacrylamide gel . A technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies. Immunoblotting is performed chiefly in diagnostic laboratories to identify the desirable protein antigens in complex mixtures. Other related technique include dot blot analysis, where antibodies are used to detect proteins in tissues and cells by immunostaining and enzyme-linked immunosorbent assay (ELISA).

7. CONTENTS • Tissue preparation • Gel electrophoresis • Transfer • Blocking • Detection • Analysis • Applications

8. TISSUE PREPARATION Samples may be taken from whole tissue, from cell culture, bacteria, virus or environmental samples. In most cases, samples are solid tissues. First broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes). Cells may also be broken open by one of the above mechanical methods. A combination of biochemical and mechanical techniques, including various types of filtration and centrifugation. To encourage lysis of cells and to solubilize proteins, may be employed : detergents, salts, and buffers To prevent the digestion of the sample by its own enzymes - Anti Protease and phosphatase To avoid protein denaturing-Tissue preparation is often done at cold temperatures

9. GEL ELECTROPHORESIS The most commonly used gel is polyacrylamide gels (PAG) and buffers loaded with sodium dodecyl sulfate (SDS). Western blot uses two types of agarose gel: stacking gel that is used for concentrate all proteins in one band and separating gel that allows for separating proteins according to their molecular weight. Smaller proteins migrate faster in SDS-PAGE when a voltage is applied. PAGE can separate proteins ranging from 5 to 2,000 kDa according to the uniform pore size which is controlled by the Different concentration of PAG. Typically separating gels are made in 5%, 8%, 10%, 12%or 15%. When we choose the appropriate percentage of the separating gel, we should consider the size of the target proteins. The smaller the known weight of proteins is the higher percentage of gels should be used.

10. TRANSFER After separating proteins by gel electrophoresis, proteins are moved from within the gel onto a solid support membrane to make the proteins accessible to antibody detection. The main method for transferring proteins is called electroblotting , which uses an electric field oriented perpendicular to the surface of the gel, to pull proteins out of the gel and move into the membrane. It can be done semi-dry or wet conditions, while wet conditions are usually more reliable as it is less likely dry out the gel. As shown in the left figure, the membrane is placed between the gel surface and filter. The transfer sandwich is created as follows: a fiber pad (sponge), filter papers, the gel, a membrane, filter papers, a fiber pad (sponge)

11. Blocking Blocking is an important step in the western blot to prevent antibodies from binding to the membrane non-specifically. The most commonly used typical blockers are BSA and non-fat dry milk. When the membrane is placed in the dilute solution of proteins, the proteins attach to all places in the membrane where the target proteins have not attached. In this way, the “noise” in the final product of the western blot can be reduced and result in clearer results

12. ANTIBODY INCUBATION After blocking, the primary antibody binds to target protein when the primary antibody is incubated with the membrane. The choice of a primary antibody depends on the antigen to be detected. Washing the membrane with the antibody-buffer solution is helpful for minimizing background and removes unbound antibodies. After rinsing the membrane, the membrane is exposed to the specific enzyme conjugated secondary antibody. When performing secondary antibody incubation, the labeled secondary antibody can bind to the primary antibody which has reacted with target proteins. Based on the species of the primary antibody, we can choose the appropriate secondary antibody

13. PROTEIN DETECTION & VISUALIZATION A substrate reacts with the enzyme that is bound to the secondary antibody to generate colored substance. It enables us to know the densitometry and location of the targets protein. And the size approximations are taken by comparing the proteins bands to the marker. There are several detection systems are available for protein visualization, such as colorimetric detection, chemiluminescent detection, radioactive detection, and fluorescent detection. The electro chemiluminescence (ECL) system is the most common detection method

14. APPLICATIONS To determine the size and amount of protein in given sample. Disease diagnosis: detects antibody against virus or bacteria in serum. Useful to detect defective proteins. Definitive test for Hepatitis B and Herpes. Identification of a specific protein in a complex mixture of proteins. In this method, known antigens of well-defined molecular weight are separated by SDS-PAGE and blotted onto nitrocellulose. Estimation of the size of the protein as well as the amount of protein present in the mixture. It is most widely used as a confirmatory test for the diagnosis of HIV, where this procedure is used to determine whether the patient has antibodies that react with one or more viral proteins or not.

15. DISADVANTAGES A disadvantage of western blotting (immunoblot) is that it is Time-consuming (compared to ELISA). High demand in terms of experience of the experimenter. Additionally, it requires optimizing the experimental conditions ( eg . Protein isolation, buffers, type of separation, gel concentration, etc.)

16. Conclusion The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Western blot technique uses three elements to achieve its task of separating a specific protein from a complex: separation by size, transfer of protein to a solid support, and marking target protein using a primary and secondary antibody to visualize. Other related techniques include dot blot analysis, quantitative dot blot, immunohistochemistry and immunocytochemistry, where antibodies are used to detect proteins in tissues and cells by immunostaining, and enzyme-linked immunosorbent assay (ELISA)