Calculation Descriptions

In the Tables there are columns that refer to calculations. When these columns are part of the table, the values are automatically calculated and reported in the table. Some columns are specific to one table and some can be shared across tables. The Columns group has commands for adding and removing columns. For further instructions, select "Adding and Removing Columns" in the help section of any table type.

Below are detailed descriptions of the calculations in these columns. First, the terms used in these descriptions are defined.

Definitions of Terms

Shape — any area enclosed using the shape tools.

Pixel — the smallest area unit of an image that is measured with a single intensity value.

Signal intensity or intensity — signal counts measured in a single pixel per unit time.

Descriptions of the Calculations

Area — The area is the total number of pixels enclosed by a shape.

Bkgnd. — The background is the average intensity of the pixels selected as the background region.

Bkgnd. Std Dev — The background standard deviation is the standard deviation of all of the background pixel intensities in a single channel.

Max — The maximum intensity is the highest pixel intensity within a shape. The maximum intensity value does not have background intensity subtracted.

Mean — The mean is the sum of all of the pixel intensities for a shape divided by the total number of pixels in a shape.

Min — The minimum intensity is the lowest pixel intensity within a shape. The minimum intensity value does not have background intensity subtracted.

Signal — The signal is the sum of the individual pixel intensity values (Total) for a shape minus the product of the average intensity values of the pixels in the background (Bkgnd) and the total number of pixels enclosed by the shape (Area). Signal = Total - (Bkgnd. x Area)

StdDev — The standard deviation of all of the pixel intensities for a shape.

Total — The total intensity is the sum of the individual pixel intensities for a shape.

Trim Bkgnd. — The trimmed background is the average intensity of the pixels selected as the background region.

The highest 5% count and lowest 5% count of the intensity pixel values of the shape are discarded.

Trim Bkgnd. StdDev — The trimmed background standard deviation is the standard deviation of all of the background pixel intensities in a single channel. Note: the highest 5% count and lowest 5% count of the intensity pixel values of the background are discarded.

Trim Mean — The trimmed mean is the sum of all of the pixel intensities for a shape divided by the total number of pixels in a shape.

The highest 5% count and lowest 5% count of the intensity pixel values of the shape are discarded.

Trim Signal — The trimmed signal is the sum of the individual pixel intensity values for a shape minus the product of the average intensity values of the pixels in the background (Bkgnd.) and the total number of pixels enclosed by the shape. Note: The highest 5% count and lowest 5% count are excluded from the intensity summation and the pixel count.

Trim StdDev — The trimmed standard deviation is the standard deviation of all of the pixel intensities for a shape.

The highest 5% count and lowest 5% count of the intensity pixel values of the shape are discarded.

In-Cell Western Calculations

Background — The average pixel intensity of each background well will be calculated, and the background wells will be averaged to find the background value to be subtracted from wells designated Sample and 100% Standard.

Linked Wells — The pixel intensities from linked wells are averaged, and this average is used in the following calculations for each of the linked wells.

ICW Relative — After background subtraction, all wells designated as Sample or 100% Standard in a channel are divided by the intensity value of the well with the highest response. This well is given a value of 1.0, and the relative intensity values of the other wells will generally be between 0.0 and 1.0.

Negative relative intensities indicate the original intensity value was lower than the average background when the background was subtracted.

If a normalization channel is selected, the intensity value of each well in the other channel is divided by the relative intensity value of the corresponding well in the normalization channel.

ICW % Response — After background subtraction and normalization (if used), all wells designated as Sample are divided by the average of the wells designated as 100% Standard in that channel and multiplied by 100 to give a percentage response to the control in the 100% Standard.

All ICW % Response values for the normalization channel are reported as NaN.

ICW Std Dev — The ICW standard deviation is the standard deviation of the average of the pixel intensities from linked wells.

Molecular Weight Estimation

First, the vertical peak location is found for all bands (MW marker bands and sample bands). This peak location is the Y value used for MW estimation. If there is a single MW marker lane, band size is interpolated based on the Y location of bands in the single marker lane. If there are multiple marker lanes, lines of constant MW across the membrane are determined by connecting bands of the same MW.

Z-Factor Calculation

First, pixel intensities in all wells designated as Background in each channel are averaged, and the average is subtracted from the pixel intensities of all wells in that channel. Then, the relative intensity values are found by dividing all wells by the intensity value of the well with the highest response. The intensity values from the sample channel are divided by the relative intensity values from the corresponding wells in the normalization channel.

The Z-factor calculation uses these values in the following equation: