255 - Frederiksen

EDVO-Kit #255Purification & Size Determination of gfp & bfpPurification & Size Determination of Green & Blue Fluorescent ProteinsBackground Informationintroduced in a protein to determine the impact of the mutation on structureand function of the protein. The set of gfp and bfp proteins can beused as a dramatic tool to visually demonstrate the effect of pivotal aminoacid changes on the structure and function of proteins. Amino acid substitutioncan also be used to demonstrate the effects of accumulated mutationson aging and various cancers.In this experiment, bacterial extracts containing gfp or bfp will be fractionatedby chromatography using a molecular sieve matrix. Factors that affectthe separation include size, shape and associated non-protein biologicalssuch as carbohydrate residues. The fluorescent proteins will be detected onthe column and subsequently in the test tubes by examination under longU.V. light. For most proteins, such columns can also be used to determineapparent molecular weights. Accurate estimation of protein polypeptidecomposition and size(s) are performed by analyzing the fractions that containthe protein of interest by denaturing polyacrylamide gel electrophoresisDuplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratoryuse only. This document, or any part, may not be reproduced or distributed for any other purpose without thewritten consent of EDVOTEK, Inc. Copyright © 1998, 2000, 2001, 2005, 2007 EDVOTEK, Inc., all rights reservedEVT 005097KThe Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com