Determining Percent Purity
From Bridges Lab Protocols
- Scan image and save as a tiff file (8-bit not 16-bit) with lanes oriented vertically.
- Open ImageJ either locally or via the webstart applet at http://rsbweb.nih.gov/ij/applets.html
- Using a rectangular box (box tool)select entire lane.
- Select lane either by selecting Analyze->Gels->Select First Lane or CTRL-1
- Plot lane by selecting Analyze->Gels->Plot Lanes or CTRL-3
- Draw baseline using the line tool.The baseline is to remove the "noise" from the blue-ness of the scanned gel. This is the reason for washing the gel with ddH20. The baseline should begin at the zero determined by where the plotted curve meets the axis.
- If necessary use the line tool to connect peaks to the baseline. Use the line tool to connect the peaks to the baseline as they would if the peaks were individual and not connected in a line. Isolate individual peaks which protrude significantly from the baseline.
- Select peaks using the wand (tracing) tool. Click on selected peaks (all peaks which are above the baseline) in order.
- Go to the results window and the areas are calculated in numerical order. Be sure to count the peaks on the plot as to determine which peak corresponds to the peak of interest.
- Copy the results into an excel file and using the sum function, sum the areas.
- Calculate purity by the area under the peak of interest divided by the area under all peaks combined.