255 - Frederiksen

Purification & Size Determination of gfp & bfpEDVO-Kit #25513Electrophoresis of ProteinsLoading Protein SamplesFine Tip Micropipet Tips(EDVOTEK® Cat. #638)are recommended forloading samples into polyacrylamidegels. A regularmicropipet tip may damagethe cassette and result inthe loss of protein samples.1. Change pipet tips between loading each sample. Make sure the wellsare cleared of all practice loading solution. Gently squirt electrophoresisbuffer into the wells with a transfer pipet.2. Two student groups can share a gel. Some of the samples contain denaturingsolution which contains SDS and 2-mercaptoethanol. The samplesshould be loaded in the following manner:First Student GroupLane 1 20µl of standard protein markers (boiled for 5 minutes)Lane 2 20µl of gfp native (not boiled)Lane 3 20µl of gfp denatured (boiled for 5 minutes)Lane 4 20µl of bfp native (not boiled)Lane 5 20µl of bfp denatured (boiled for 5 minutes)The ExperimentSecond Student GroupLane 6 20µl of standard protein markers (boiled for 5 minutes)Lane 7 20µl of gfp native (not boiled)Lane 8 20µl of gfp denatured (boiled for 5 minutes)Lane 9 20µl of bfp native (not boiled)Lane 10 20µl of bfp denatured(boiled for 5 minutes)NOTE:Shine the long wave U.V.light on the gel whilethe native proteins areseparating.Running the Gel3. After the samples are loaded, carefully snap the cover all the way downonto the electrode terminals. On EDVOTEK® electrophoresis units, theblack lead on the cover should be aligned with the terminal identifiedby the black dot.4. Insert the black lead into the black input of the power supply (negativeinput). Insert the red lead into the red input of the power supply (positiveinput).Time and VoltageVoltsRecommended TimeMinimum Optimal125 40 min 1.0 hrs70 60 min 1.5 hrs5. Set the power supply at the required voltage and run theelectrophoresis for the length of time as determined byyour instructor. When the current is flowing, you shouldsee bubbles forming on the electrodes. The sudsing is dueto the SDS in the buffer.6. After the electrophoresis is finished, turn off power, unplugthe unit, disconnect the leads and remove the cover.Duplication 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