Electrophoresis Simulation How To Page


Getting Started

  1. If the ELECTROPHORESIS PARAMETERS & ELECTROPHORESIS CELL panels are displayed advance to Setting Simulation Conditions.
  2. Click on the Set Parameters button. Experimental simulation parameters are set in this panel.
  3. Click on the Simulation button. This panel is a representation of two wells in a slab gel electrophoresis cell.

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Setting Simulation Conditions

  1. Select an animation speed. This option was provided to allow simulation speed adjustment based on your computer's capability.
  2. Unknown #1 is default.You can use unknown #1 or [Optional] select 1 of the 10 unknowns from the drop down box.
  3. 7.5% Acrylamide is default. [Optional] Select 1 of the 4 concentrations. Increasing the % acrylamide will result in smaller pore sizes in the gel. The smaller pore sizes retard the migration of all the proteins in the gel; larger protein are more affected than smaller proteins.
  4. Select 2 or more standards by clicking the check box next to the name. You can see the full name of the protein and its molecular weight by having the Check out Protein Information for additional information on each standard.
  5. 100 Volts is default. [Optional] Select 1 of the 4 voltages. Increasing the voltage will increase protein elution speeds.

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Protein Information

  1. Click on the Protein Info button. The PROTEIN DATA panel displays select information on the currently selected protein.
  2. Selected a protein on the PARAMETERS panel. The data panel will be updated with information about the selected protein.

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Run Simulation

  1. Insure that the PARAMETERS & ELECTROPHORESIS CELL panels are displayed. Click on the Set Parameters & Simulation buttons if necessary.
  2. Two or more standards should be selected.
  3. Click on the Add Standard button. A simulation of the standard addition will run.
  4. Click on the Add Sample button. A simulation of the sample addition will run.
  5. Click on the Start Run button. The protein bands will elute down the cell and separate based on molecular weight.
  6. Click on Stop Run before the first band, the dye band reaches the bottom of the cell.

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Get Data on the Protein Bands

  1. Click on any of the bands in the cell and the relative migration (RM) will be displayed at the top of the cell panel.
  2. Click on the band from the unknown or sample side of the cell. The RM of the unknown will be displayed.

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Plot of Standard Results

  1. Click on the Plot Results button. A plot of the log molecular weight (Log MW) vs relative migration (RM) will be displayed.
  2. The line fitted through the standards RM results will be displayed with its slope, y intercept and goodness of fit statistics.

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Plotting the Unknown

  1. Slide the mouse pointer along the RM axis until the displayed RM position is approximately the same as the unknown's RM.
  2. Left click and a line will be plotted from the RM axis to the fitted line and over to the Log MW axis.
  3. The experimental Log MW will be displayed near the axis and the Experimental MW will be calculated and displayed above the graph.
  4. If the Experimental MW is close to the unknowns actual molecular weight the unknowns identity will be displayed.
  5. If you get a message indicating the RM was close but the unknown's identity was not, check for a poor line fit. Another possible cause is a % Acrylamide value that was too high to allow the unknown to elute properly.

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Goodness of Fit Statistics

  1. r Squared is an indication of the "goodness of fit" or how strong the coorelation was between molecular weight and relative migration.
  2. A r Squared value of unity or 1 is ideal. Lower values indicate less correlation.
  3. A possible cause for low coorelation values is a % Acrylamide that is too high for the standands chosen.

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dmix@kodak.com.
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