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Western blotting


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  • 1. Presented By MADIHA AHMAD 1
  • 2. Blots are techniques for transferring DNA , RNA and proteins onto a carrier so they can be separated, and often follows the use of a gel electrophoresis. The Southern blot is used for transferring DNA, the Northern blot for RNA and the western blot for PROTEIN 2
  • 3. w 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 3
  • 4. A technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies. 4
  • 5. Tissue preparation Gel electrophoresis Transfer Blocking Detection Analysis 5
  • 6. Samples may be taken from_ whole tissue or from cell culture. In most cases, solid tissues are first broken down mechanically using a blender. _It should be noted that bacteria, virus or environmental samples can be the source of protein and thus Western blotting is not restricted to cellular studies only. Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins. Tissue preparation is often done at cold temperatures to avoid protein denaturing 6
  • 7. The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point molecular weight, electric charge, or a combination of these factors. 7
  • 8. 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). 8
  • 9. 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. Another method for transferring the proteins is called electro blotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane 9
  • 10. The membrane has the ability to bind to proteins in in this case both the target and antibodies are proteins and so there could be some unwanted binding. Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein - typically Bovine serum albumin(BSA) with a minute percentage of detergent such as Tween 20. 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. 10
  • 11. There are two steps for the detection of the protein 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 The solution is composed of buffered saline solution with a small percentage of detergent, and sometimes with powdered milk. The antibody solution and the membrane can be sealed and incubated together for anywhere from 30 minutes to overnight 11
  • 12. 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. Several secondary antibodies will bind to one primary antibody and enhance the signal 12
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  • 14. calorimetric detection The colorimetric detection method depends on incubation of the western blot with a substrate that reacts with the reporter enzyme (such as peroxidase) Chemiluminescent detection Chemiluminescent detection methods depend on incubation of the western blot with a substrate that will luminescent when exposed to the reporter on the secondary antibody. 14
  • 15.  Radioactive detection Radioactive labels do not require enzyme substrates, but rather allow the placement of medical X-ray film directly against the western blot which develops as it is exposed to the label and creates dark regions which correspond to the protein bands of interest 15
  • 16. •The confirmatory HIV test employs a western blot to detect anti-HIV antibody in a human serum sample. Proteins from known HIV-infected cells are separated and blotted on a membrane as above. Then, the serum to be tested is applied in the primary antibody incubation step; free antibody is washed away, and a secondary anti-human antibody linked to an enzyme signal is added. The stained bands then indicate the proteins to which the patient's serum contains antibody. Western blot can also be used as a confirmatory test for Hepatitis B infection. 16
  • 17. If a protein is degraded quickly, Western blotting won't detect it well. This test takes longer that other existing tests It might also be more costly 17
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