Primary and Secondary Antibodies Western blotting is typically performed by prob

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Primary and Secondary Antibodies Western blotting is typically performed by probing the blocked membrane with a primary antibody that recognizes a specific protein. The choice of a primary antibody for a Western blot will depend on the antigen to be detected and what antibodies are available to that antigen. It is also important to note that not all primary antibodies are suitable for Western blotting and the feature should be verified, if possible, before purchasing a new primary antibody. In general, the primary antibody which recognizes the target protein in a Western blot is not directly detectable. Therefore, tagged secondary antibodies or other detection reagents are used as the means of ultimately detecting the target antigen (indirect detection). A wide variety of labeled secondary detection reagents can be used for Western blot detection. The choice of which depends upon either the species of animal in which the primary antibody was raised (the host species) or any tag on that antibody (i.e., usually an enzyme). For example, if the primary antibody is an unmodified mouse monoclonal antibody then the secondary antibody must be an anti-mouse IgG secondary antibody obtained from a non-mouse host. Antibodies for Western blotting are typically used as dilute solutions, ranging from a 1/100- 1/500,000 dilution from a 1 mg/ml stock solution. The optimal dilution of a given antibody with a particular detection system must be determined experimentally. More sensitive detection systems require less antibody than lower sensitivity systems and can result in substantial savings on antibody costs and allow a limited supply of antibody to be stretched out over more experiments. Using lower amounts of antibody can also have the added benefit of reduced background because the limited amount of antibody shows increased specificity for the target with the highest affinity Antibody dilutions can be made in the wash buffer. However, the presence of detergent and a small amount of the blocking agent in the antibody diluent often helps to minimize background thereby increasing the sianal:noise ratio. Hence, the antibody dilutions can be made in bocking buffer Detection Methods While there are many different tags that can be conjugated to a secondary or primary antibody the detection method used will limit the choice of what can be used in a Westerm blotting assav. Radioisotopes were used extensivelv.in the oast. but thev are expensive. have a E Focus +10

Explanation / Answer

Ans 5- alpha tubulin mouse monoclonal antibody- 1:1000-1:5000 dilution for western blotting analysis.

Rabbit antimouse IgG secondary antibody- 1:400 dilution conjugated with fluorescent dye.

Beta actin polyclonal antibody 1:100-1:2000 dilution.

Goat antirabbit IgG H&L HRP conjugated. 1:400 dilution.

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