"EXTRAS"

Here is cut material that may be incorporated elsewhere.

A great deal more basic knowledge must be accumulated in order to understand and construct or reconstruct living tissues from the "bottom up." We won't obtain such knowledge by pursuing only those research topics that have immediate, or for that matter foreseeable, benefits. Basic, or pure, research seeks to discover all there is to know about nature, pursuing avenues that are most likely to reveal something of significance.

You should be aware now of the importance of simple biological models to the understanding of cellular processes in more complex systems.

The study of a well known system is a good starting point for exploring the features of less well characterized systems.

All cells have a cytoskeleton, and share common elements and modes of organization. Since the cytoskeleton is intimately involved in cell function, including cell-cell interactions, receptor organization, motility, organization of organelles, endocytosis and exocytosis, cell division, etc., it is extremely important to understand the cytoskeletal organization of cells. The mammalian erythrocyte is probably the easiest of all cells to use as a model for the organization of cytoskeletal proteins, and in fact the first detailed models of cytoskeletal structure were developed for the erythrocyte membrane. Blood is easily obtained from either animals or humans. The red cells are easily separated from other components by washing and centrifugation. The cells themselves are easily taken apart by osmotic shock and the membranes separated, again by washing and centrifugation. Very pure samples of membrane with associated intact cytoskeleton can be obtained using fairly simple techniques.

In order to study organisms, organs, tissues, and cells we have to take them apart. The disruption of cells and purification of components is itself an applied science. Ideally, components that are separated will retain both their structure and function insofar as they can function independently of other cellular structures. That can be particularly difficult, especially when enzyme function requires membrane integrity, since the membrane must be disrupted in order to begin cell fractionation in the first place. To begin to learn some principles of cell fraction and to obtain good material for study, it is best to start with the simplest model obtainable. The mammalian erythrocyte (red blood cell) serves that purpose well.

We conduct a simple fractionation in the teaching lab in order to obtain fairly pure preparations of red cell (erythrocyte) plasma membranes, with their associated proteins. Homogenization is not necessary, since whole blood treated with anticoagulant remains a liquid. We first use centrifugation to separate blood plasma from the formed elements (red and white cells, and platelets). We then lyse the red cells and separate the membranes from the cytoplasm.

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.

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.

[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."

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.

More details on the Bradford assay are reported elsewhere. Note that the description is generalized - the information in the protein assay problem describes how we will conduct the assay in the teaching lab. 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.

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.

In an early form of electrophoresis, disolved protein mixtures were placed in a U-shaped buffer-filled channel and subjected to an electric field. Resolution was poor and any disturbance of the apparatus compromised the separation. Gels were developed to serve as solid supports for electrophoresis, so that the separated products remain separated and can be easily stained and handled. The development of the stacking gel, which compresses the sample into bands a few micrometers thick, added a major improvement to the resolution of gels (Ornstein, 1964; Davis, 1964). Other landmark improvements to protein electrophoresis were the use of polyacrylamide for control of separation by molecular size, and the use of sodium dodecyl sulfate (SDS; lauryl sulfate) to denature proteins in order to ensure reproducibility of the technique (Weber and Osborn, 1969; U.K. Laemmli, 1970).

A separating gel of given acrylamide concentration separates proteins effectively within a characteristic range. The largest polypeptides can enter a low percentage gel readily, and are fairly well separated. However, such a gel has a relatively low cutoff. That is, polypeptides below a particluar size are not restricted at all by the gel, and all move at the same pace, along with the tracking dye, regardless of size. A gel of 7% acrylamide composition typically has a cutoff of 45 kiloDaltons. A gel of very high percentage acrylamide may restrict all of the proteins in a mixture. The smallest protein of any significance among the fractions of mammalian blood is hemoglobin (14 kD). The hemoglobin band is readily resolved in a 15% acrylamide gel, but is buried in the dye front in a 7% gel. The problem with running just a 15% gel is that the heavier proteins are so restricted that they are jammed near the top of the gel and are not easily resolved from one another. In fact, it is a good idea to forget about analyzing the top third of such a gel. To take advantage of the characteristics of both low and high percentage gels, we usually run both.

In the teaching lab we recommend that alternate teams prepare low or high percent gels, with each team exchanging samples with a team that prepared the other type gel. Each team, then, would load its set of samples, appropriate standards, and another team's samples on its gel, and have its samples loaded onto another percent gel as well. In addition to expanding the range of resolution of bands, this practice allows comparison between identical fractions prepared by different teams, to control for inconsistencies in fractionation, sample preparation, etc.

Interpretation of an SDS gel is usually straightforward. The investigator looks for a particular band or pattern of bands that are characteristic of the biological sample that was collected. The gel is examined to determine purity of the preparation or the presence/absence/modification of specific protein bands. SDS-PAGE can be combined with specialized techniques such as immunoblotting, two-dimensional electrophoresis, peptide mapping, etc. in order to identify specific proteins or protein isoforms (two or more versions of the same protein with slightly altered structure).

The relative mobility of a polypeptide in SDS-PAGE is typically related directly to the log of its molecular weight. However many factors act to modify the migration of individual polypeptides, so that the molecular weight determined from a gel is seldom identical to the molecular weight that would be obtained from the actual amino acid sequence. Nonprotein substituents such as carbohydrate or lipid residues can exercise 'drag' on the polypeptide. Denaturation may be incomplete for polypeptides with long stretches of hydrophobic residues, which is true of many transmembrane proteins. A protein may have a large proportion of acidic or basic residues, which alter its charge-to-mass ratio. Despite one's best efforts, the protein may suffer some degradation, leading to a faster rate of migration than for the intact polypeptide.

The current model for the structure of the red blood cell membrane should be used as a guide to identification, along with other sources of information. The molecular masses of the known proteins have been established. The number corresponding to molecular mass is identical to that corresponding to molecular weight (e.g., molecular mass of 200 kDa corresponds to molecular weight of 200,000). Apparent molecular weights are only one basis for identification, and should not be relied upon solely. Also consider quantity of protein (indicated by intensity of a band), the fraction(s) in which a band is found, associations with other bands, and quality of a band. As tentative identifications are made, one must consider the uncertainty inherent in the technique. Some proteins may not show up at all, because they are present in too few numbers or they don't stain with the method used.

As mentioned previously, 'nonprotein' residues on polypeptide chains can influence migration, leading to indistinct bands and deviation of apparent molecular weight from the true molecular weight. Proteins with a high carbohydrate content are notorious for migrating with unpredictable relative mobilities, and for failing to stain with standard methods. Species differences and differences in method of determination of published molecular weights can also lead to disparities between an estimate and published values.

SDS-PAGE is used for a variety of applications, mostly involved with monitoring the purity of protein fractions and for identification of specific proteins. Western blotting, two-dimensional electrophoresis, and peptide mapping are among techniques used for verification of the identity of specific proteins. Such techniques are also used to study unknown proteins, to determine if they are identical to, have any structure in common with, known proteins.

Even without conducting the specialized tests of identity, one can often characterize the SDS-PAGE profile of a sample based on information already known about the biological system that is studied. Such initial charactererization provides a basis for further study by allowing the investigator to ensure that samples are pure and not degraded. It also provides a basis on which the investigator can apply specialized techniques for identification of isoforms, products of genetically engineered mutant genes, expression of known proteins in different cell types, etc.

first examine the gels and label all of the bands that were resolved, starting with the major (darkest and densest) bands. Be aware that it may take more than one gel to see the complete profile, depending on the molecular weight 'cut-offs.' In higher density gels the bands at the top may be compressed so that what appears to be one band is really two or more bands.

Use molecular weight standards to construct a standard curve, and estimate apparent molecular weights for the bands you have noted. Keep in mind that the relationship between relative mobility and molecular weight is logarithmic, so that it is necessary to round your determinations to a reasonable number of significant digits. What is the smallest dimension that can be resolved on the gel? What molecular weight range does that dimension span? Be careful, since the molecular weight range spanned by a given dimension depends on where you are on the gel. Use that range to determine how to round your results. In any event, you certainly cannot estimate apparent molecular weights to the nearest unit or even ten units.

Prepare a table of descriptive characteristics of the bands you have labeled, including apparent molecular weight, relative intensity of the band, extent to which the band is resolved, and associations with other bands. You can use relative terminology of course - dark/light, thick/thin, distinct/'fuzzy,' forms a doublet with band ---, etc..

Once you have characterized your unknowns, you need to consult literature for information on the proteins known to be part of the system you are studying. For example, if you are analyzing an erythrocye membrane fraction, look for papers that present the current model of red cell membrane structure. Consider the quantity of each known protein that you would expect to find in the fraction, known molecular weight of the protein, whether or not it is likely to be water-soluble or is a transmembrane protein, and whether or not isoforms of the same protein are common. Find candidates for the known protein among the bands you described. In some cases, identification may be so straightforward that you are fairly certain of a band's identity. In other cases, you may have no clue as to what you have.

Use molecular weight standards to construct a standard curve, and estimate apparent molecular weights for the bands you have noted. Keep in mind that the relationship between relative mobility and molecular weight is logarithmic, so that it is necessary to round your determinations to a reasonable number of significant digits. What is the smallest dimension that can be resolved on the gel? What molecular weight range does that dimension span? Be careful, since the molecular weight range spanned by a given dimension depends on where you are on the gel. Use that range to determine how to round your results. In any event, you certainly cannot estimate apparent molecular weights to the nearest unit or even ten units.