UVP Doc-It Life Science - Manual (Page 91)

Performing 1D Analysis
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Retardation factor (Rf) Lines
Automatic Rf Line Determination
When two or more lines are calibrated with molecular weight standards, LS Software creates Retardation
factor (Rf) Lines automatically. These lines express any differences in horizontal alignment between
bands (or points on a lane) of equal molecular weight. Ideally, there will be one Rf line for each distinct
molecular weight used in a calibration. Since users may utilize more than one standard on a single
image, and each standard may contain several weights, automatic generation can result in a large
number of Rf lines.
An automatic RF line can be moved by re-calibrating the lanes. An automatic Rf line can be adjusted by
dragging the lane-intercept marker up or down, but only in lanes that are not calibrated.
To Adjust an Automatic Rf Line
If Rf lines are not visible, turn them on through Analysis > 1D Analysis > Settings. When the
Preferences window opens, select the 1D Analysis tab and click on the Rf line box (so that a
check appears in the box) in the Visible 1D analysis objects area.
Select the Rf line to adjust. Control handles will appear.
Any lane that has a white control handle without an "X" is an uncalibrated lane, and it is possible
to move the intercept point up or down by dragging it. If a lane has a gray control handle with an
"X," it is calibrated and cannot be moved.
After adjusting the Rf lines, hide them to view the other 1D objects more easily. To hide the Rf
lines, go to Analysis > 1D Analysis > Settings. When the Preferences window opens, select
the 1D Analysis Settings and unclick Rf line in the Visible 1D analysis objects area.
Adding Rf Lines Manually
LS Software allows users to add Rf lines to an image with less than two lanes that are calibrated to
molecular weight standards. (On images with two or more calibrated lanes, Rf lines are created
automatically and the software will not allow new lines to be added, although the automatically added
lines can be adjusted as described in the Automatic Rf Line Determination section.)
To add an Rf line manually
On the Analysis > 1D Analysis > Rf lines, click Add Rf Line or click on Add RF line in the 1D
Analysis Plugin. Rf lines will be made visible if they were formerly hidden, and a window will pop
up entitled Add Rf Line.
The cursor will now appear as a square cross. Select the first band that is part of the Rf
relationship in the captured image.
Click on a second and subsequent bands in other different lanes. Click on as many bands as
preferred to draw the line (up to one per lane), but at least two must be chosen.
To place an Rf line anchor (circle) on a non-band location, hold down the CONTROL (CTRL) key
as the circle is placed.
When finished selecting the points that will make up the line, click Confirm Add in the Add Rf
Line dialog window. The green line will now appear white, and a new Rf line will appear.
The software will require the user to select a standard lane. Identify the standard lane by clicking
on the standard lane in the image. The standard lane will be outlined in green when selected.
To place more Rf lines, follow the same process. Note, however, that new Rf lines are allowed as
long as they do not cross another Rf line. If the line appears green, the Rf line is allowed. If there