You perform a western-press-blot o detect albumin from rat serum. You are convin

Question

You perform a western-press-blot o 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 do 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 into a rat. To detect Nesprin-1 human cells in a western-blot press which HRP (horse radish peroxidase)-conjugated secondary antibody would you use? rat anti-rabbit rat anti-mouse

Explanation / Answer

6. option b is correct. Close contact of gel and membrane is necessary to ensure a clear image during transfer. After separating the protein mixture (gel), it is transferred to a membrane. If you will not press this membrane and gel (protein mixture) properly, then proteins will not move out of the gel onto membrane. Hence there will be no reaction between protein and antibodies and thus the colour of all the band will remain same.

If you will forget to add the chloronapthol then no purple colour will produce at all while here it is mentioned that the purple colour development for each band hence option a is not possible.

Gelatin is used as blocking agent in western blotting test which block binding of antibodies to membrane. If you will gorget it then the antibodies will bind with membrane but with protein also. Hence option c is also not possible.

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