Question

In an experiment to isolate the lipids from horse red blood cells, one of the steps is too "Add 500µL of 10mM sodium phosphate buffer (pH. 7.0). Resuspend the pellet thoroughly by vortexing."

What happens in this step of the lipid isolation period? (hint: the supernatant in the next centrifuge step will be red)

Answer 1

RBCs (Red Blood Cells) are biconcave discs present in the blood and contains large amounts of hemoglobin. They assist in transport of O2 and CO2, clearance of immune complexes, etc.

RBCs also serve as a good model to study plasma membrane and cytoskeleton particularly because they do not contain a nucleus or organelles and so the plasma membrane and its associated proteins can be easily isolated. they also do not possess microtubules and intermediate filament.

The plasma membrane contains 2 transmembrane proteins -

Glycophorin

Band 3

A network of spectrin tetramers associate with the plasma membrane connected by stretches of actin filaments. Protein 4.1 binds to spectrin and actin filament. Ankyrin mediates the conatct of spectrin network with the plasma membrane.

In the above question, suring RBC isolation to isolate lipids, sodium phosphate is used.

Sodium phosphate solution isotonic to the RBCs is used to wash the RBC pellet. This step removes any additional plasma components attached to the RBCs and allows us to get a pure RBC pellet.

However, since the question states that the supernatant becomes red, it means that the sodium phosphate solution used was hypotonic and resulted in bursting of RBCs to release its content (which is hemoglobin mostly). Since, the solution was hypotonic, water moved from the external solution to inside the cell due to which the cell swelled and eventually burst. Thus the supernatant becomes red.

This would leave a nearly white pellet at the bottom. It would become completly white upon 2-3 times washing.