Overview: Characterization of red cell membrane proteins by SDS-PAGE

Analysis of SDS-PAGE

The 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).

An objective of this part of the study is to learn some of the strategies for recognizing protein bands and patterns on a gel. Another objective is to learn the limitations of the technique - identification of a band on a protein gel is not considered positive proof of identity. One more objective is to learn to improve one's performance by recognizing the consequences of imperfect technique, and the disasterous consequences of major errors in gel/sample preparation and running gels.

Image the gel

We used to have to take photographs of gels for analysis, scan them on densitometers, or, if we were really desparate, photocopy the gel directly. Now that high quality digital images can be routinely made, scanning and digitizing a gel image is the most convenient way of preparing an image for analysis. An important original gel should be dried down and saved.

Critique your gels

Examine each of the gels on which your samples have been run, and using the poster materials (see the guide) evaluate your performance. If something went wrong, the "hall of shame" should help you identify the cause.

Identify gels, protein lanes, and obtain relative mobilities (Rfs)

As silly as it sounds, it is important to identify which gel is which (percent acrylamide and which samples were loaded), top and bottom of each gel, and the lanes corresponding to specific samples. All of that information, including identifying marks and labels, should be recorded in the notebook.

The top of the gel is the top of the separating gel. The stacker is of no use now and can be removed. If the dye front is not visible, either it ran off the gel or is not visible because no proteins ran with the tracking dye (usually with high percent acrylamide gels). Each band of importance should be labeled (without obscuring information on the image) and a relative mobility determined for each, using either the dye front or, if the gel has calibrated standards, the bottom of the gel as a reference. Relative mobility is determined by measuring the distance from the top of the gel to the middle of the dye front or arbitrary reference point, measuring the distance from the top of the gel to the middle of the band, and dividing the second measurement from the first. This is the Rf, which is always between 0 and 1.

Note that the relative mobility of a given protein depends on gel concentration. Any single gel has an upper and lower limit to its useful range for estimating molecular weights.

Prepare a molecular weight standard curve

The relationship between relative mobility and molecular weight is logarithmic, in a perfect gel of uniform concentration. Your results will always be a bit less than perfect, however a standard curve will turn out nearly linear when a log scale is used. On semilog paper, or using a graphics program with y-axis on a log scale, the molecular weight of each calibrated standard should be plotted on the y-axis, versus its Rf on the x-axis.

Obtain apparent molecular weights

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 molecular weight as determined from SDS-PAGE is called an apparent molecular weight. Often it is close to the true molecular weight of the polypeptide, but sometimes the apparent molecular weight can be way off - as is the case for one or more erythrocyte membrane proteins. By the way, molecular weight and molecular mass are quantitatively identical, but molecular weight is a relative term thus the quantity is dimensionless.

The position of each unknown band relative to the protein standards should be noted before using the standard curve. The standards may not fall exactly on the curve fit even though they were specifically chosen because their apparent molecular weights are very close to their true molecular weights. A curve fit with a straight line may not intersect the topmost or bottommost standard. Because the relationship is logarithmic, an error at the bottom is no big deal. However an error at the top of the gel can result in a huge over- or underestimation. It might be a good idea just to connect the data points and interpolate apparent molecular weight directly, ignoring the 'curve.'

The molecular weight that corresponds to the Rf of the polypeptide band is read from the standard, and recorded with a reasonable number of significant figures. Precision can be estimated from the minimum resolution on a gel (usually ~0.5 mm) and the molecular weight range covered by that distance at that position on the gel (each gel is more accurate toward the bottom than toward the top).

Characterize the membrane-associated proteins

See the topic "identifying bands on protein gels" for a general approach to characterizing the protein composition of a fraction.

SDS-PAGE of purified organelles such as plasma membranes, ribosomes, endoplasmic reticulum, etc. usually gives patterns that are dominated by one or a few major protein bands. On a 7 to 8% gel, erythrocyte membranes yield a pattern that is dominated by: the heavy (>200 kd) spectrin bands, which form a doublet at the top; the wide anion exchanger protein band (band 3) in the range of 100 kd or so; and the actin band at around 43 kd. An investigator should look for the pattern, double check that the apparent molecular weights are in the right ball park, and use the basic pattern as a framework for identification of less dense bands.

If the pattern is not apparent, there may have been a problem with the fractionation, sample preparation, or the gel. The results should be compared with illustrations showing electrophoresis of red cell membrane preparations, and the procedures double checked if the pattern isn't at least somewhat close to the typical pattern. Degradation of proteins or failure of the reducing agent are common reasons for such 'strange' results.

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.



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Created by David R. Caprette (caprette@rice.edu), Rice UniversityDates