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Seperation and detection of proteins 1. What was the purpose ofwashing the red blood cells with saline prior to lysis? 2. Whatproperty of the lysis buffer caused the red blood cells to lyse? 3.What are the differences between the primary and secondaryantibodies used in this study? 4. What are the advantages of usingindirect immunodetection over direct immunodetection? 5. Antibodiesare made when animals detect a foreign antigen. All mammals carryalbumin in their blood. How was a rabbit antibody against albumincreated? Was this lethal to the rabbit? Explain you reasoning. 6.Albumin is not a cytoskeletal protein. What was the purpose ofusing antibodies to detect albumin in our cytoskeletalpreparations? 7. Why was it necessary to transfer the proteinsseparated by SDS-PAGE onto nitrocellulose membranes prior toincubation with anti-albumin antibodies? 8. Why did weelectrophoretically transfer the proteins resolved by SDS-PAGE ontonitrocellulose? Can you suggest another way to transfer proteinsfrom a SDS gel to a membrane? 9. Why did we wash the nitrocelluloseblots with blotting buffer three times after incubating with theprimary and secondary antibodies?
The rate of enzyme activity at varying temperatures:
If you had brought the test tube that had been incubation at 0 degrees Celsius back to 37 degrees Celsius, what change in the reaction rate would you expect to see? Why?
If you had brought the test tube that had been incubation at 80 degrees Celsius back to 37 degrees Celsius, what change in the reaction rate would you expect to see? Why?
The purpose of the alkaline phosphatase reaction as part of theWestern blot procedure is to
a. determine the number and purity of target bands on thepolyacrylamide gel.
b. help visualize specific proteins at particular locations onthe nitrocellulose membrane.
c. degrade target proteins, releasing a colored phosphorylatedproduct of the Western blot.
d. bind and react with primary and secondary antibodies,removing a phosphate.
e. locate the immunoglobulins on the Western blot that werepresent in the cell extract.
Blocking solution
a. prevents non-specific binding of antibodies to thenitrocellulose membrane.
b. prevents binding of primary and secondary antibodies whilethey are incubating together.
c. inhibits the activity of cellular enzymes, especiallyproteases, during the incubation.
d. is used to wash out unbound primary antibodies before thesecondary antibody solution is added to nitrocellulose.
e. improves binding of immunoglobulins to the nitrocellulosemembrane.
