Isolation and characterization of erythrocyte membranes

Isolation and characterization of erythrocyte membranes

Objective:

·         To be familiar with protein and lipid isolation protocols.

·         To study about the composition of human red blood cell membrane. 

Introduction:

            In this experiment the membrane constituent of the red blood cells (erythrocytes) membrane was isolated. Protein and lipid isolation form biological systems are important process in biochemical researches. Generally proteins are made with amino acid monomers which have some polarity so these proteins can dissolve in water or ethanol like solvents. Although lipids are somewhat different than proteins which are consist of more hydrophobic properties than proteins therefore lipids are more favorable to dissolve in nonpolar solvents. So using those qualities the whole extraction procedure is conducted.   

            Erythrocytes are commonly use cell type in this practical because it can be obtained easily and also it has not organelles so it would not contain RNA and DNA other than that this cells conduct relatively little metabolism and the offer a homogeneous source of easily obtainable membranes.

Erythrocytes consist of three basic components. Those are lipid bilayer, trans-membrane proteins and a cytoskeletal network. The lipid bilayer is a semipermeable layer which is asymmetric in composition and separates the cytoplasm from the extracellular medium. Phosphatidylcholine, sphingomyelin, phosphatidylserine, phosphatidylethanolamine and the sterol cholesterol are the dominant component of it.

Transmembrane proteins are immerged in the bilayer, and those proteins have great mobility in the plane of the membrane. The major transmembrane proteins are glygoproteins, band 3 and glygophorin. Band 3 is a multispanning ion transport channel. Other than that Ankyrin, Glycophorins , Aquaporins (water channel proteins) and Rh (is a protein complex) are present in the membrane as proteins. The cytoskeleton is an irregular hexagonal lattice of fibrous polypeptide called spectrin, which are tied together by actin. The skeleton makes a two dimensional network which is very flexible and compressible. 

Centrifugation technique was highly used throughout this experiment. Centrifugation is a separation of a heterogeneous mixture to different layers by spinning it. A centrifuge is the equipment that use in this technique. This equipment is generally driven by an electric motor, which puts an object in rotation around a fixed axis, applying a force perpendicular to the axis to separate substances of different densities. Using this phenomena blood samples can be separated to layers as the cells and the plasma.  

Throughout the extraction procedure well buffered system should be maintained. This buffer maintained the isotonic solution for erythrocytes and it prevents the lysis of the cells. This is done because in this experiment the erythrocyte membrane should be isolated not the whole proteins in blood. so the to do that the plasma should be removed from the samples to isolate the erythrocytes, plasma also containing some proteins like Albumins, Globulins, Fibrinogen so those proteins must not be in the erythrocyte membrane extract. But after the isolation of erythrocytes has been done the cells would be lysis using a hypotonic solution.

While this experiment is conducting samples should be maintained at 4 ºC temperature because to reduce the enzymatic activities. All the cells have compartments which contains hydrolytic enzymes that are capable of breaking down all kinds of biomolecules, including proteins, nucleic acids, carbohydrates and lipids. But in this experiment the whole membrane should be isolated without getting denatured or disrupted of the protein/lipid constituents of it.

Methodology

Materials: Micro centrifuge tubes, centrifuge, vortex mixture micro pipettes, test tubes, droppers

Reagents: blood sample, 310 mM and 20 mM sodium phosphate buffer, chloroform, chloroform: methanol mixture (1:2), methanol, 0.1 M HCl, distilled water.

a.       Isolation of erythrocyte membranes from human blood.

ü  Maintain 4 ºC temperatures throughout this protocol.

To 10 ml of blood sample,

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Add 1.78 ml of 310 mM sodium phosphate buffer and EDTA,

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Mix and centrifuge at 1000g for 10 min,

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Remove the buffer coat using micro pipette,

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Resuspend the pallet in 6.67 ml of buffer,

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Mix and centrifuge (at 4 ºC) at 1000g for 10 min (above 3 steps repeat 4 times),

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Resuspend the above pallet with buffer to 1.67 ml of final volume,

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Add 0.278 ml of above suspension to 1.39 ml of 10mM (hypotonic) phosphate buffer,

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Invert several times to mix,

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Centrifuge (at 4 ºC) at 14000g for 40 min,

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Remove the supernatant,

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Resuspend the pallet in 1.39 ml of 10mM (hypotonic) phosphate buffer,

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Centrifuge (at 4 ºC) at 14000g for 20 min,

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Repeat the above 3 steps for 2 more times,

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Remove the supernatant and get the loosely packed membrane pallet.

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The residue is saved for further experiments.

b.      Lipid extraction

Get 0.044 ml of above final extraction,

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Add 0.167 ml of chloroform: methanol (1:2) mixture,

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Mix vigorously with vortex mixture for 30 sec,

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Add 0.055m ml of chloroform,

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Mix vigorously with vortex mixture for 30 sec,

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Add 0.055 ml of distilled water,

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Mix vigorously with vortex mixture for 30 sec,

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Centrifuge at 500g for 5 min,

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(Denatured proteins stuck in the chloroform; water interface. If the protein layer extends to the bottom layer add 0.0055 ml of 0.1 M HCl and mix and centrifuge again)

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Remove as much as upper methanol/water layer,

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Using a syringe carefully puncture thought the protein layer,

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Then get the bottom chloroform layer to clean weighted tube,

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Evaporate the Separated chloroform layer (use hot water bath).

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                              The residue is saved for further experiments. 

Discussion

            Blood samples were obtained in different donors who are in different genders. All the samples were mixed with anticoagulant because blood can be clotted outside of the body and if the blood was clotted the cell isolation process should be very difficult. Usually Heparin sulfate will use as an anticoagulant but in this lab session EDTA was used. The EDTA can make complexes with metal ions as a chelator. Ca²⁺ ions are needed to coagulation process so when EDTA was used the Ca²⁺ ions can be removed from the solution and prevented the coagulation.

After the first centrifugation step the expected color of the supernatant was pale yellow color because after centrifugation the cells and plasma would be separated. But the observed color was reddish yellow color this happens because of some red blood cells has been lysed when the buffer was added, the Tonicity of the blood cells was not known exactly. The isotonic buffer was prepared as it was given according the previous experiment results in lab manuals. The tonicity of blood can be differ with the genetic trait as well as the donors feeding habits (usual water intake).  

           Throughout the membrane extraction procedure all the mixing or dissolutions were conduct in gently manner and the vortex mixer was not used in this procedure. Because the vigorous swirls can damage and destruct the protein molecules. So this can cause false observation in the SDS-PAGE separation experiment.

In the lipid extraction procedure chloroform methanol water system was used, because most of lipids have non polar properties as it was mentioned earlier. But membrane-associated lipids are more polar and require polar solvents such as methanol to disrupt hydrogen bondings or electrostatic forces. So using a methanol chloroform mixture is more efficient to extract lipids, and chloroform can denature proteins as well.