Seperation and detection of proteins 1. What was the purpose of washing the red

Question

Seperation and detection of proteins 1. What was the purpose of washing the red blood cells with saline prior to lysis? 2. What property of the lysis buffer caused the red blood cells to lyse? 3. What are the differences between the primary and secondary antibodies used in this study? 4. What are the advantages of using indirect immunodetection over direct immunodetection? 5. Antibodies are made when animals detect a foreign antigen. All mammals carry albumin in their blood. How was a rabbit antibody against albumin created? Was this lethal to the rabbit? Explain you reasoning. 6. Albumin is not a cytoskeletal protein. What was the purpose of using antibodies to detect albumin in our cytoskeletal preparations? 7. Why was it necessary to transfer the proteins separated by SDS-PAGE onto nitrocellulose membranes prior to incubation with anti-albumin antibodies? 8. Why did we electrophoretically transfer the proteins resolved by SDS-PAGE onto nitrocellulose? Can you suggest another way to transfer proteins from a SDS gel to a membrane? 9. Why did we wash the nitrocellulose blots with blotting buffer three times after incubating with the primary and secondary antibodies?

Explanation / Answer

1. Washing the red blood cells with saline removes plasma and traces of buffy coat. Saline also protects the red blood cells from bursting due to osmosis.

2. RBC lysis buffer increases the influx of ions inside the cells, which results in the build-up of osmotic pressure inside the cell. So, water flow into the cell, which results in the cell lysis.

3. Primary antibodies are used to bind the RBC proteins, while secondary antibodies containing fluorescent label bind the primary antibodies.

4. Indirect immunodetection provides more amplification that occurs by using a secondary antibody.

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