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Western Blotting

Western Blotting

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Western Blotting

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  1. Western Blotting Corey Lyons Stevens Institute of Technology Harlem Children’s Society

  2. Who discovered this technique? • This Technique was discovered by Dr. Douglas Lake of the University of Arizona School of Medicine's Department of Microbiology and Immunology. • Dr. Lake is now studying how the immune system can be harnessed to attack cancer.

  3. What is Western Blotting? • A technique in which proteins are separated by gel electrophoresis and transferred to a membrane sheet. A specific protein is then identified through its reaction with a labeled antibody.

  4. Steps in a Western Blot • The first step is gel electrophoresis.(The proteins of the sample are separated according to size on a gel.) • The second is Membrane Transfer.(The proteins in the gel are then transferred onto a membrane made of nitrocellulose by applying current.) • The third step is Blocking.(Blocking is used to prevent non-specific protein interactions between the membrane and the antibody protein.)

  5. Interesting Fact • When Blocking in Western Blotting the solution most used is Carnations Non Fat Dry Milk. • No one knows why Carnation’s works best.

  6. Incubation • Incubation occurs by diluting an antibody into a solution which will keep the pH neutral. The majority of the time the solution is non fat dry milk. • The Primary antibody is then incubated into the membrane. • The primary antibody recognizes only the protein of interest, and will not bind any of the other proteins on the membrane.

  7. Incubation Continued • After rinsing the membrane to remove unbound primary antibody a secondary antibody is incubated with the membrane. • It binds to the first antibody. • This secondary antibody is usually linked to an enzyme that can allow for visual identification of where on the membrane it has bound. • Finally, the reaction product may produce enough fluorescence to be detected on a sensitive sheet of film when it is placed against the membrane.

  8. Where is this technique used? • The HIV Test known as "Western Blot" uses the same technique, where the goal is to detect the presence of antibody in a sample. Known HIV infected cells are opened and their proteins separated and blotted on a membrane. Then the serum to be tested is applied. Free antibody is washed away, and a secondary antibody is added that binds to human antibody and is linked to an enzyme. The stained bands then indicates the proteins to which the patient's serum contains antibody.

  9. Conclusion • I believe that with Western Blotting money and time will be saved for people suffering from HIV. • The virus will be detected faster and the proper treatment will be used for the patients specific need.

  10. Acknowledgments • The Harlem Children’s Society • Dr. Sat Bhattacharya • The Stevens Institute of Technology • Professor Saxena