You perform a western press blot to detect albumin from rat serum You are convin

Question

You perform a western press blot to detect albumin from rat serum You are convinced that you did every single step of the protocol perfectly. Yet, when you did the color development reaction you find that all the protein bands in every single sample turned purple. Thus you did not get a specific reaction for albumin Which step you think you did wrong? You forgot to add the chloronapthol in your color development solution You did not press hard enough to allow transfer of proteins to the nitrocellulose membrane You forgot to add Gelatin All of the above You have generated an antibody to a human protein called Nesprin-1 by injecting purified human Nesprin-1 into a rat To detect Nesprin-1 from human cells in western-blot press which HRP (horse radish peroxidase)- conjugated secondary antibody would you use? rat anti-rabbi rat anti-mouse rabbit-anti rat mouse anti-rabbi. Which of the following is true about lectins? Lectins are carbohydrate binding proteins Lectins play roles in cell signaling and cell-cell interactions Receptors for lectins are cell-surface glycoproteins All of the above

Explanation / Answer

6. c) You forgot to add Gelatin

Explanation: In western blotting, blocking buffer is used to block unoccupied binding sites on the support to specifically detect the protein immobilized on solid support. Blocking may be accomplished by a variety of protein and detergent solutions; among which Gelatin Blocking Buffer is the common as it is compatible with a variety of detection methods. Target molecules can be immobilized on nitrocellulose, nylon, and charge-modified nylon membranes.

As we forgot to add gelatin, therefore all the protein bands in every single sample turned purple because, chloronaphthol will bind to total protein onto the nitrocellulose or other blotting membranes and substrate will develop an insoluble, purple product on the membrane surface after exposure to horseradish peroxidase conjugated antibodies.

7. c) rabbit anti rat HRP conjugated secondary antibody will be used.

8. d) All of the above.

Explanation: Lectins are specific carbohydrate binding proteins that are ubiquitous and found in animals, plants and microorganisms. The exact role of lectins in plants is unclear, however, they can serve as potent insecticides. The main function of lectins in animals is to facilitate cell-cell contact which is achieved by the binding sites. The binding sites of lectins on the surface of one cell interact with arrays of carbohydrates displayed on the surface of another cell. Therefore lectins play an important role in cell signalling and sell cell interactions. Receptors for the lectins are cell surface glycoproteins.

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