Western%20Blotting

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Western Blotting

• Corey Lyons
• Stevens Institute of Technology
• Harlem Childrens Society

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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.

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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.

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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.)

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Interesting Fact

• When Blocking in Western Blotting the solution
  most used is Carnations Non Fat Dry Milk.
• No one knows why Carnations works best.

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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.

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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.

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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.

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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.

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Acknowledgments

• The Harlem Childrens Society
• Dr. Sat Bhattacharya
• The Stevens Institute of Technology
• Professor Saxena