# Blot Quantification

## Calculation Definitions

• Area: Number of pixels enclosed by a lane box or band box.

• Signal: Sum of the individual pixel intensity values (Total) for a band box (or lane box) minus the product of the background value and area.

Signal = Total - (Background x Area)

• Pixel Intensity: A value proportional to the amount of light collected from a single pixel. You can adjust the overall brightness of an image, but pixel intensities remains the same.

• Total: Sum of the individual pixel intensities enclosed by a band box or lane box.

## Quantification of Total Protein in a Lane

### Signal

Sum of the individual pixel intensity values (Total) for a lane minus the product of the background value and area.

Signal = Total - (Background x Area)

### Average Signal ### Background

Empiria Studio 1.3 automatically evaluates the local area around each band box or lane box to determine the background value. Read this section for more information about how Adaptive Background Subtraction works.

If the computed background value is extremely small, a "0" will appear in the Analysis Table in the Background column. To see all the digits of the computed background value, export the Analysis Table to a CSV file or to an Excel spreadsheet.

### St. Dev.

Standard deviation of lane signals within a replicate group. n = The number of data points

xi = Each of the values of the data

x̅ = The mean of the xi

### % CV

Coefficient of variation of signals for lanes within a replicate group. ### SNR

The signal-to-noise ratio (SNR) compares the signal in a lane to the background noise calculated for the lane. See Signal-to-Noise Ratio for more information.

### Avg. SNR

Average signal-to-noise ratio for lanes within a replicate group.

## Band Quantification

### Signal

Sum of the individual pixel intensity values (Total) for a band minus the product of the background value and area.

Signal = Total - (Background x Area)

### Avg. Norm. Signal

The average of the normalized signal values for target bands in a replicate group.

## Background

Empiria Studio 1.3 automatically evaluates the local area around each band box or lane box to determine the background value. Read this section for more information about how Adaptive Background Subtraction works.

If the computed background value is extremely small, a "0" will appear in the Analysis Table in the Background column. To see all the digits of the computed background value, export the Analysis Table to a CSV file or to an Excel spreadsheet.

### SNR

The signal-to-noise ratio (SNR) compares the signal for a band to the background value calculated for that band. See Signal-to-Noise Ratio for more information.

### Avg. SNR

The average signal-to-noise ratio for target signals within a replicate group.

### Std. Dev.

Standard deviation of target signals within a replicate group. n = The number of data points

xi = Each of the values of the data

x̅ = The mean of the xi

### % CV

Coefficient of variation for signals for target signals within a replicate group. ### Fold Change ## Normalization

1. A lane normalization factor is calculated based on the internal loading control Endogenous protein(s) that are unaffected by experimental conditions and used as an indicator of sample loading. band or lane with the highest signal. 2. The normalized signal for each band is calculated by dividing the signal for each band by the Lane Normalization factor for the lane the band is in. ## Signal-to-Noise Ratio

The signal-to-noise ratio (SNR) compares the signal Sum of the individual pixel intensity values (Total) for a quantification shape minus the background value. in a quantification shape (e.g. band box, well circle) to the background value calculated for that shape.

### Calculating the Signal-to-Noise Ratio

In Empiria Studio, the SNR is calculated using this formula: ### Interpreting the Signal-to-Noise Ratio

The following points are helpful to keep in mind when interpreting the SNR for your analysis. Data interpretation is specific to your experiment, and it may be best to consult a statistician.

• If your SNR is less than 1, ensure your lane boundary includes enough lane area around the bands. If insufficient lane area is included, the shapes assigned to your bands may be too large and this may lower the SNR. If this occurs, adjust the size of shapes around your bands as needed.

• By convention, an SNR ≥ 3 is considered a reliable threshold for distinguishing signal from background noise.