This App fits a standard curve to relate the known molecular weights of proteins in a set of standards (y-axis) to their relative migration (x-axis), and estimates molecular weights of unknow proteins.
Download the file "GelMWAnalyzer.opx", and then drag-and-drop onto the Origin workspace. An icon will appear in the Apps gallery window.
- Click the App icon to load an image and a dialog with 4 panels is opened.
Note: Lanes in the image should be vertical and are scanned from the top to the bottom.
- There are two steps in the determination of molecular weight.
- Step 1: Define a lane of standard sample and fit a standard curve.
- In the upper left panel, draw a rectangle on the image to define a lane that contains the standard proteins.
- In the upper right panel, detect or add bands that mark the positions of the standard proteins in the profile.
- In the bottom left panel, enter know molecular weight into each row of bands.
- Select function from linear, polynomial and exponential decay. Click Fit Standard Curve to fit a curve of Log10(MW) vs Rf (or migration distance). The fitted curve is shown in the bottom right panel.
- Click Next button.
- Step 2: Define a lane of unknown sample and calculate molecular weight.
- Upper left panel:
- Light on Dark: If the spots on the image are lighter than the background, check this option to correctly display the profile.
- Add New Lane: Draw a rectangle on the image to add a new lane.
- Modify Lanes: Click the button to make the lanes resizable and draggable.
- Delete Selected Lane: Click a lane to select it and then click the button to delete it.
- Align: Align the top of selected lane to the top of the 1st lane. In this way, you can ensure different lanes start at the same position.
- Upper right panel:
- Detect Bands: Detect bands and mark their positions.
Note: You can click the gear icon beside the button to modify the search window's width and height which are used to detect bands. For example, if two bands are too close to distinguish, you can reduce search window size.
- Add New Band: Add a new band manually.
- Delete Selected Band: Click a band to select it and then click the button to delete it.
- Delete All Bands: Delete all bands marked on the profile.
- Bottom left panel:
- Convert X to Relative Migration: Treat last band as dye front to calculate relative migration (Rf). Fit a standard curve of Log10(MW) vs Rf. Otherwise fit a standard curve of Log10(MW) vs X.
- Fit Standard Curve: Fit a standard curve from the standard sample.
- Calculate Molecular Weight: Calculate molecular weight of unknown sample by the regression equation.
This app provides a sample image. Right click on the app icon in the Apps Gallery window, and choose Show Samples Folder from the short-cut menu. A folder will open. Open the Example.pdf file that shows detailed steps to calculate molecular weight with the sample image. The output report is contained in Gel sample.opju.