Recordkeeping, Writing,
& Data Analysis


Microscope studies

Flagella experiment
Laboratory math
Blood fractionation
Gel electrophoresis
Protein gel analysis
Concepts/ theory
Keeping a lab notebook
Writing research papers
Dimensions & units
Using figures (graphs)
Examples of graphs
Experimental error
Representing error
Applying statistics
Principles of microscopy

Solutions & dilutions
Protein assays
Fractionation & centrifugation
Radioisotopes and detection

Characterizing Bands on Protein Gels

One should have an idea of what to expect when running a gel, including what proteins will be separated and should make use of any available information about their constituent polypeptides. For a well characterized system such as mamalian eryhrocyte membranes, a good cell biology textbook should present the current model including relative abundance and molecular masses for prominent membrane associated proteins. Less well studied systems may require research into the primary literature. The nature of one's research determines how much information is needed and/or how much is available. Seldom if ever should an investigator be faced with characterizing a sample of completely unknown composition.

A good approach to characterizing a sample is to seek the identities of the most prominent bands first, that is, those that stain most intensely. A few solid identifications can provide a framework within which to deal with fainter bands if it is necessary to consider them at all. If similar samples have been previously characterized then one might also look on the gel for bands with masses corresponding to those of polypeptides expected to be in the sample.

Characterization efforts should consider all of the evidence, including molecular mass (or MW, relative molecular mass), qualitative observations on the bands, and distribution of bands into recognizable patterns. It is valuable to consider the known structure and function of a candidate protein, not only when conducting a characterization but also when defending a conclusion in a discussion. For example, serum albumin is the most abundant single protein in blood plasma. It is soluble and has molecular mass of around 66 kDa in most mammalian species. The presence of a well resolved thick, dark band with apparent mass of 66 kDa suggests that it represents serum albumin.

Keep in mind that what you determine from a gel are apparent molecular weights, which will deviate from the true molecular weights, sometimes by quite a bit. Finally, keep in mind that you have denatured the proteins - the native forms may consist of multiple subunits and be associated with nonprotein molecules. An example is hemoglobin, which in native form consists of four subunits associated with a heme group. Published values for the molecular weight of hemoblobin may be for the native form, while on your gels the most prominent hemoglobin band will be of the monomer.

When you have difficulty finding a polypeptide that you know should be present, consider that many polypeptides do not run true to their known molecular masses, and may simply be out of place on the gel. Two polypeptides of similar mass may not resolve, but form a single band instead. A very dark, thick may mask the presence of a fainter band with migration range that overlaps that of a more abundant polypeptide. Some polypeptides simply are not stained well by Coomassie Blue dye. Such is true of the red cell membrane-associated protein glycophorin. Carbohydrate residues bound to the protein repel the stain. Other stains such as periodic acid-Schiff (PAS) reveal glycophorin. There is another complication, though. The same residues that repel the stain also exert "drag" on the molecules, causing the 25 kDa glycophorin subunit to migrate as if it had a mass of 100 kDa.

Some bands may represent products of degradation of a heavier polypeptide. Unless there is massive deterioration within a sample such bands will be lighter than the originals. Bands may represent unseparated subunits, that is, two or more polypeptides that remain attached to each other, as can happen with hemoglobin. Some bands may simply be aggregates of nonspecifically bound polypeptides. Aggregated material sometimes appears at the very top of a lane.

Finally, there is the possibility that you have stumbled upon a novel (previously undescribed) protein, especially if you are analyzing a sample from an uncommonly used species. Maybe you've found a research project!

Copyright and Intended Use
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Created by David R. Caprette (caprette@rice.edu), Rice University 9 Oct 96
Updated 18 Nov 12