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We have developed a simple and inexpensive experiment using gel filtration chromatography that can be performed in less than 90 minutes. This procedure not only provides students with hands-on experience in both the preparation and use of chromatography columns, but also uses colored molecules to provide dramatic visual confirmation of the process of size-exclusion chromatography. Additionally, the column can be calibrated using molecules of known molecular weights and can then be used to estimate the molecular weights of unknown samples. Our gel filtration experiment costs only a fraction of that of commercially marketed laboratory exercises. A basic understanding of methods and techniques in modern life science research is important in understanding the way that life scientists develop new methods for answering biological questions and the potentials and limitations of these modern experimental methods. On both the introductory college and high school levels, many students are performing basic exercises using electrophoresis and recombinant DNA. Unfortunately, few of these students are exposed to chromatography, the major means of purifying proteins and other biological molecules. This is partly because of the expense and complexity of many chromatographic procedures. Our experiment involves a simplified version of the analytical methods used by research laboratories to fractionate proteins and estimate molecular weights by gel filtration. This exercise was modified from such procedures performed by one of the authors during his postdoctoral training. This experiment illustrates the concepts and mechanisms of gel filtration and gives a simplified understanding of both chromatography column preparation and typical data analysis. It is currently used as a lab exercise in the introductory Cell Biology course at Jacksonville State University. Our experiment offers significant advantages over commercially marketed chromatography experiments. These kits often provide pre-packed columns while our protocol allows students to actually prepare the column. Additionally, many commercial gel filtration experiments require the use of an expensive, ultraviolet-capable spectrophotometer to monitor the elution profile, while our experiment requires only an inexpensive visible light spectrometer. Finally, our experiment costs less than a dollar per student (after an initial purchase of columns and gel filtration media) compared with the $10 per student cost through commercial competitors. Although initial purchases of needed supplies will cost slightly over a hundred dollars, reagent and material costs for repeated exercises are very low. The hardware may be rinsed and reused, and the Sephadex can be re-used as well.

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The American Biology Teacher, 2000, 62(8), 602–607.

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Biology Commons



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