TotalLab Quant FAQ

• Why does Automatic Lane Creation fail on my gel?
• How does Molecular Sizing work?
• Why use different curve types?
• What is the name of the curve fitting algorithm?
• My image does not look like the image I captured. Why?
• My profile is upside down. What can I do to fix it?
• What file formats can the software support?
• When I load in my JPG file the image is inverted
• I cannot load my tiff image?
• What is Band Volume?
• Where can you get the total volume of all the bands in a Lane?
• What is the background level in Array and why don't all spots have the same Background Volume?
• What is Profile Deconvolution?
• How do I turn Profile Deconvolution on?

Why does Automatic Lane Creation fail on my gel?

There could be a number of reasons this happens. It could be because

• The lanes are short or have very few bands
• The lanes are too close together so the algorithm cannot split them
• The lanes are not quite the same width which is an assumption the algorithm uses
• The resolution of the image is very low and there is not enough band information

Using an AOI for lane detection can help (just draw a box when in automatic detection mode) or simply try to create the lanes manually.

How does Molecular Sizing work?

Each lane has its own calibration curve calculated from where the iso-molecular weight lines(yellow) cross the centre of a lane. With only one standard lane these curves are usually the same as the lines just run horizontally across the gel. However with more than one standard lane it joins up the bands of the same value and so the lines can be slightly off horizontal depending on the gel.

Why use different curve types?

The default curve fit is cubic spline. This curve type creates a smooth curve passing through all points. It has no defining equation. This option can be somewhat over sensitive to inaccurate mappings but will guarantee that the results are as expected from where the iso-molecular weight lines(yellow) cross the lanes. First order Lagrange is similar but uses straight lines.

The other main option used historically is the Linear Log equation which is a straight line calculated from the log of the Molecular Size. This is theoretically correct but can lead to misleading results due to any inaccuracy of the curve fit. Even curves with high R2 results can still have problems.

What is the name of the curve fitting algorithm?

We use an iterative algorithm called Levenberg-Marquardt to get the results for the curve fitting equations. Cubic Spline and First order Lagrange are also well defined algorithms.

My image does not look like the image I captured. Why?

A histogram of all pixel values is created. Then the default auto contrast is created from the value of the histogram after 0.5% of all pixels and at 99.5% of all pixels. This stops outliers or scanner noise affecting the result but it can look a little different

If the image has a calibration (from Powerscan) or an encoding (from GE or Fuji scanners) then the converted values are used by default for the image. If you do not want this then use the "Raw" option to go back to the raw pixels values

Note: Contrast does not affect the values used in any calculations

My profile is upside down. What can I do to fix it?

Go to the File menu and select Invert Measurements to reverse the calculation used to generate the Profile. This option is remembered so you should not need to change it again.

What file formats can the software support?

The software can load in .tif, .tiff, .jpg,.jpeg, .png, .gif, .gel, .img (Fuji), .bmp files.

When I load in my JPG file the image is inverted

This occasionally happens as part of the loading function. Just press the invert button on the toolbar to see if reversed again.

I cannot load my tiff image?

One type of tiff image we cannot load is a multi-page tiff. These are very rare in life science and are only used for items such as multi page faxes. There are many free tools which can split the tiff into single image files including ImageMagick.

What is Band Volume?

This is defined as the total value of all the pixels in a band with background subtracted. The pixels are shown on the image as between the 2 red lines (the band edges). Another way to look at it is as the area under the profile for the band (with background subtracted) multiplied by the lane width.

Where can you get the total volume of all the bands in a Lane?

This information can be found in the Analysis Report (previously called Gel Report). On the first page total band volume (and total lane volume) are displayed in a summary table of all the lanes in the gel.

What is the background level in Array and why don't all spots have the same Background Volume?

The background level is the value calculated from the selected method as the background used for that spot. It can be the same for all spots. The value is subtracted from every pixel in the spot and then all pixels are summed to get the spot volume. If some pixels in the spot are lower than the background level then they are only set to zero and not a negative number. Therefore you can get differing background totals, though not by that much usually.

What is Profile Deconvolution?

This option allows the user to model each band as a gaussian and use these to recalculate the band volume. This is sometimes used to split the volume of overlapping bands in what is believed to be a more accurate manner. Advanced fitting is best but there are problems. They are firstly, that is can be very slow and secondly, without good background subtraction they can be very wrong.

How do I turn Profile Deconvolution on?

By default in TotalLab Quant this option is off. You can turn the option on from the start or finish pages of the wizard by ticking the check box next to Profile Deconvolution.