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Steps of the Western blot analysis Essay Example

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Steps of the Western blot analysis

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The third step entails gel electrophoresis. In order to separate the different proteins from each other, the lysades became loaded on SDS polyacrylamide gel. SDS as a component of the gel allowed for neutralization of the proteins thereby making their separations entirely based on molecular weight (Kontermann 2002, p. The separation became performed by use of constant voltage. In order to visualize, the use of a dye called bromophenol blue became used (Rosenberg 2005, p.The fourth step in western blot analysis involved blotting. In here, blotting refers to the direct transfer of the given proteins to the membrane from the gel by use of electric power plus nitrocellulose. The membrane pores of nitrocellulose permanently accommodate the given proteins transfer from the gel (Rosenberg 2005, p.More over, the fifth step in western blotting analysis involves blocking. This process involves filling with empty pores of the given nitrocellulose membrane with some certain proteins that will not be recognized by antibodies used commonly in the experiment (BSCL bovine serum albumin) (Rosenberg 2005, p.The sixth step entails incubation with primary antibody. In here, blocked membranes become incubated in a solution containing primary antibody that got the capability of recognizing target proteins. Normally, the primary antibody often becomes dissolved in the given buffer used for deblocking. The primary antibody often permanently binds to the given target protein (Rosenberg 2005, p.The seventh step in western blot analysis entails application of the secondary antibody. Here, a secondary antibody becomes applied after primary antibody incubation. The secondary antibody often goes and binds to the primary antibody forming a protein-antibody-antibody sand witch. Normally, the secondary antibody becomes labeled with florescent dye for the purpose of visibility (Rosenberg 2005, p.Therefore, the eighth procedure in western blot analysis involves visualization. Here, the visualization of the target protein and antibodies become possible. This could be achieved by the help of fluorescent scanning. Usually, the secondary antibody contain peroxidase enzyme that often converts luminal substrate to give a light releasing substrate. It hence becomes the light which becomes detected as a spot on the film (Rosenberg 2005, p.In the ninth step, in western blot

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Bibliography

King, D., 2003, Applications and engineering of monoclonal antibodies, New York: Taylor &

Francis.

Kontermann, R., 2002, Antibody Engineering, New York: Springer.

Michael Stumpf, D. J., 2011, Handbook of Statistical Systems Biology, New York: John Wiley

and Sons.

Pound, J., 2001, Immunochemical Protocols, Massachussets: Springer.

Rosenberg, I. M., 2005, Protein analysis and purification: benchtop techniques, New York:

Springer.

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