Blood and Erythrocyte Fractionation

The term fractionation refers to dismantling cells or tissues and separating components, so that single components can be analyzed in the absence of contaminants that might change a result and/or mislead an investigator. Fractionation protocols are designed for specific applications, however most such protocols share two common features, namely disruption of tissues and/or cells to release their components, and differential centrifugation to separate major categories of components. A plethora of separation methods have been developed for further fractionation in order to obtain specific cell types, organelles, or macromolecules, but these are the most common starting points.

The unique structure of blood makes it very easy to separate red blood cells from plasma and the other formed elements by differential centrifugation. Once isolated, red cells can be lysed (burst open) by suspension in a hypotonic medium. They take up water by osmosis and eventually explode, leaving an empty membrane sack (sometimes called a "ghost") behind.

Before conducting a fractionation for the first time, you should review the principles behind tissue fractionation, collection of samples, and determination of yields.

Materials

When whole blood is collected from a mammalian source it must be treated with anticoagulant and either used immediately or stored refrigerated until use. It is necessary to record the species and to record the source if the strain of animal might make a difference. The purpose of the fractionation dictates the volume of blood that should be used. You would need a very large volume, for example, if you were to try to purify a specific protein (other than hemoglobin, which is present in very high concentrations in red cells). If the intention is strictly qualitative analysis, such as characterization of one or more fractions by SDS-PAGE, then a small volume, say 2 or 3 ml, will do.

A suitable isotonic (iso-osmotic) buffer consists of 0.9% NaCl with 5 mM sodium phosphate, pH 8. A suitable hypotonic buffer consists of 5 mM sodium phosphate, pH 8, without NaCl. Distilled water alone would be fine, but you would have no control over pH. If the procedure is conducted at ice bucket temperature and is carried out fairly quickly, no preservative such as sodium azide should be needed.

Red cell isolation

We have had good results by conducting the fractionation directly in centrifuge tubes that hold three or so volumes of buffer in addition to the original whole blood sample. One volume refers to the volume of sample. Thus if the volume of whole blood is three milliliters, three volumes of isotonic buffer would be 9 ml. Any fractionation procedure results in losses due to incomplete separation of components, materials sticking to surfaces, etc. The smaller the final pellet, the more likely too much of it will be lost through attrition. We can fit 13 ml into our Sorvall SM-24 centrifuge tubes without spilliage during centrifugation, so we typically start with two or three ml of whole blood and bring the volume to the 13 ml mark with isotonic buffer. The13 ml level is best marked on the tubes in advance.

A buffer that is considered to be isotonic for a cell type has the same osmolarity as the environment in which the cell is normally found. Thus it neither swells nor shrinks (crenates) in the buffer. The major electrolyte in blood plasma as well as in the interstitial fluid in vertebrates is sodium choride, so the logical choice for an isotonic buffer is 0.9% NaCl. It is necessary to mix the sample thoroughly with buffer to begin the process of washing the red blood cells free of plasma proteins. An effective means of mixing is trituration. Trituration refers to repeatedly pulling liquid into a pipet and ejecting it, while keeping the tip immersed. The suspension should be centrifuged immediately following trituration. Spinning down the cells separates the cell pellet from most of the plasma proteins, which are soluble and remain in the supernatant. Cell pellets are seldom tightly packed, however, so without one more wash there may still be plasma proteins in with the pellet.

Centrifugation at 600 x g brings down the red cells quickly. The low speed works because the cells are loaded with hemoglobin, an iron-containing protein. Ten minutes is more than enough time to separate red cell pellet from dilute plasma supernatant. After removing an aliquot of supernatant the remaining liquid should be discarded and the pellet resuspended in isotonic buffer (to the 13 ml mark) by trituration, then re-centrifuged. To monitor the efficiency of separation it is only necessary to remove fractions following the initial separation of supernatant and pellet, not from the wash steps.

Lysis and recovery of membranes

After two "spins," the buffy layer containing white blood cells should be lost, and platelets will not have spun down as quickly as red cells, so the pellet should consist almost exclusively of red blood cells. Resuspension in hypotonic buffer by trituration lyses most of the cells. Lysis can be confirmed by watching the suspension as the pellet is mixed. Whole red cells produce a cloudy, opaque suspension. Once lysed, the suspension turns clear although it will remain a dark red. Lysis releases the red cell contents (almost entirely hemoglobin) into the buffer, leaving empty membrane sacks, or red cell "ghosts" in suspension.

[Aside - a suspension is a heterogeneous system consisting of fine particles mixed into a liquid. Particles in a typical suspension eventually settle out if undisturbed, forming a sediment at the bottom of the container. A solution, on the other hand, is a homogeneous system in which molecules of solute are evenly dispersed throughout a liquid solvent, interacting with the solvent in such a way that they remain dispersed even if undisturbed. Cell membranes form a suspension. Hemoglobin is a soluble protein, thus the hemoglobin released from lysed red cells is in solution].

The aliquot of lysed red cell suspension should be kept small, since the greater the volume of the aliquot, the less the membrane yield. Red cell membranes pellet quickly when centrifuged at 12,000 x g. The process again takes about ten minutes. The supernatant, consisting of dilute cytoplasm, primarily contains hemoglobin. The membrane pellet, which is difficult to see due to the red color of the supernatant, will be quite red and must be washed several times in order to remove most of the remaining hemoglobin. After taking an aliquot of supernatant the pellet should be triturated in hypotonic buffer and recentrifuged, washing one or more additional times until the pellet is nearly white or at least a much lighter red color.

Membranes do not pack tightly, thus the volume of the membrane pellet may be close to that of the original whole blood sample. A small fibrous mass will appear following each high speed spin. It consists of clotted material (fibrin, platelets, and both red and white blood cells) and is not readily dispersed. It can be ignored until it is time to recover the membrane fraction, at which time it is most efficient simply to gently agitate the tube in order to liquify the membrane pellet, then remove the pellet to a sample tube using a micropipettor, leaving the fibrous mass stuck to the centrifuge tube. If it does get loose, it is easily removed using a wooden applicator stick.

Notes on fractionation

• Experienced lab personnel NEVER stick pipets or anything else into a stock bottle. They estimate how much they will need, and pour a working amount into a small container, from which they pipet what they need. Experienced personnel never return such material to a stock bottle. This practice helps protect stocks from contamination.
• Cold iso-osmotic buffer at pH 8 maintains cellular integrity until it is time to lyse them. The alkaline pH and cold both prevent decomposition of the membranes. Obviously, holding the tube by the bottom is likely to warm it up, as is leaving it out of the ice bucket for a long period of time.
• The quicker you accomplish the fractionation the better the results. Letting the preparation sit at your bench while you figure out what to do next is not recommended.
• The object of centrifugation is to separate components, a purpose that is defeated if they are re-mixed while pipetting the supernatant. Never eject liquid from a pipet near the surface of a pellet. If the pellet starts to mix, stop and re-centrifuge.
• The membrane pellet does not pack very well and is hard to see at first. It is easily pulled up and discarded by accident. To avoid losing the sample, the first supernatant following lysis should be removed with extreme care, and if the surface of the pellet can no longer be distinguished, it should be resuspended and re-centrifuged immediately. The pellet will be much more visible once the hemoglobin has been somewhat diluted. It will be necessary, in that case, to take another aliquot of dilute cytoplasm in order to obtain an accurate estimate of the yield of that fraction.
• Although the primary interest is in the membrane proteins, the collection of aliquots and electrophoresis of other fractions along with the membrane samples allows monitoring of the effectiveness of the separation procedure.
• Even well-packed pellets include a substantial liquid component, so that solutes are progressively diluted by re-suspension but not completely removed. When a solute is highly concentrated, as is hemoglobin following red cell lysis, a significant amount may remain as a contaminant even after several washes.
• The object of a wash step is to rid individual components of contaminating solutes. If chunks remain in suspension, the wash step is ineffective. Each time, the pellet must be completely dispersed.
• As lysis proceeds, ruptured cells empty their contents increasing the osmotic strength of the medium, until a balance is reached in which the medium is again isotonic with the remaining intact cells. If intact cells remain after resuspension in hypotonic buffer, they will lyse during the next wash.
• [aside] The terms "erythrocyte" and "red blood cell" can be used interchangeably. However, the phrase "erythrocyte cell" is redundant. the suffix "-cyte" means "cell." Another even more commonly used redundancy is the term "RPMs." RPM stands for "revolutions per minute," refering to the number of times a point on an object that spins on its axis passes the same spot. RPM is a plural term. If you say "RPMs," it is the same as saying "revolutions per minutes."

Protein assay

There will be plenty of time to prepare protein standards for a Bradford assay during the fractionation steps. You can also prepare aliquots for assay as they are obtained, one at a time. When the final (membrane) aliquot is obtained, it can be prepared for assay and color reagent added to all of the standards and unknowns. You should be able to finish collecting data for the protein assay within fifteen minutes or so of completing the cell fractionation.

Bovine serum albumin (BSA) is a commonly used protein standard, and is fine for our purposes even though the color reagent is about twice as sensitive to BSA as it is to many other proteins. Since an objective is to reveal as many bands as we can, it is preferable to underestimate the protein concentration (thus overloading a lane) than to overestimate it and dilute the samples too much. Using a 100 µl volume for each standard or sample, and 5 ml color reagent per sample, a typical batch of lab-prepared reagent is sensitive to between 5 and 100 µg protein. Beyond this range absorbance doesn't change sufficiently with changes in amount protein, i.e., the color reagent becomes saturated. The Bio-Rad Corporation sells a Bradford reagent concentrate which is more sensitive and more consistent. It is expensive and because the reagent contains a high concentration of phosphoric acid the shipping expense includes a steep hazardous materials charge.

It is convenient to use a stock solution of 1 mg/ml BSA for preparation of standards, diluting with either hypotonic or isotonic buffer, since neither buffer affects the results. Six to eight standards plus the reference tube should be all you need. These samples, even the membrane sample, readily dissolve in the color reagent, so it is not necessary to use sodium hydroxide to solubilize the samples.

Typical concentrations for the aliquots collected in this study are

• Plasma fraction, 4 to 10 mg/ml
• Lysate fraction, 20 to 50 mg/ml
• Cytoplasm fraction, 20 to 50 mg/ml
• Membrane fraction,1 to 8 mg/ml

Many factors affect the protein concentrations, so do not be surprised if you discover that some or all of yours fall outside of the suggested ranges.

More details on the Bradford assay are reported elsewhere. A plot of absorbance at 595 nm vs. amount protein (a standard curve) should be hand-drawn in the notebook. For convenience it can also be plotted with the aid of a computer later, although a hand-drawn curve will be sufficiently accurate. The concentration of protein in each of the four aliquots can then be estimated using the standard curve.

Notes on the assay

• One experienced individual can conduct the fractionation and protein assay in less than one hour. Two inexperienced individuals such as yourselves will take a good three hours to finish. You will save time by dividing responsibilities (but keep a complete set of notes on ALL procedures).
• As you obtain each aliquot, prepare it for the protein assay (saving the remainder on ice, of course).
• Keep the protein assay tubes at room temperature. Some breakdown will take place, but total protein content will be unaffected. If you keep the tubes on ice, they will fog up in the spectrophotometer.
• Unless an assay is well-established in a laboratory, color reagent should be added to standards and unknowns in sequence at the same time, so that they are read under the exact same conditions and in the same time frame. Even when an assay is in frequent use, at least one standard should be prepared and read in case something changes.
• Read absorbance, not transmittance. Absorbance is directly proportional to concentration.
• If both assay tubes for a sample give readings that are off scale, simply repeat the assay for that sample only, doubling or halving the amount used depending on whether there was too much or too little. The same standard curve can be used, so the repeat takes only a few minutes.
• If both assay tubes for a sample give usable readings ( on scale), then use the lower of the two absorbances to determine amount of protein. Because the absorbance scale is logarithmic, lower readings are more accurate. It is not appropriate to average the two readings, and of course only the completely clueless would report two different concentrations for the same sample.

Record keeping

You can't correct mistakes or even repeat your work if you don't recall how you did it. You can't predict what will go wrong, so the only tried and true method of troubleshooting is to keep thorough records of all procedures as you work in the lab. It is critical to record HOW everything was done, not just what was done. Much more detail is included in a lab notebook than you would report in the materials and methods section of a paper, since you may need that information to trace the history of the experiment. Here is a non-inclusive list of records that should be kept.

• How much blood did you start with? What was the source? How was it delivered to you?
• What was the type of centrifuge used? Which one did you use (in case it turns out to have been mis-calibrated)?
• Have you ever before used an automatic pipettor? Record the proper technique for pipetting as described to you in lab.
• Have you ever calibrated a spectrophotometer before? Record how to set it up, what scale was read, wavelength used.
• Not only will thorough notes help you troubleshoot a failed experiment, but you may find that as you record them, you catch mistakes before it is too late to correct them (e.g., recording absorbances at the wrong wavelength).

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