Western Total Protein Normalization

Normalize a western target band to total-lane protein and a reference lane.

Description

Western-blot normalization first relates a target band to total lane protein.

Western-blot normalization first relates a target band to total lane protein. Dividing that ratio by a reference-lane ratio expresses relative signal on the same basis.^[1]

Normalize a western target band to total-lane protein and a reference lane.

When to use Western Total Protein Normalization

Use this tool to normalize a western target band to total-lane protein and a reference lane.

Prepare the canonical sequence, peptide assignments, fitted assay parameters or upstream measurements requested. Keep corresponding entries aligned, use the stated mass and signal units, and remove FASTA headers before entering sequence text.

How to use Western Total Protein Normalization and understand its results

Prepare the canonical sequence, peptide assignments, fitted assay parameters or upstream measurements requested. Keep corresponding entries aligned, use the stated mass and signal units, and remove FASTA headers before entering sequence text.
Enter targetBandIntensity, laneTotalProteinIntensity, referenceLaneRatio using the field table and calculation rules, then run the tool.
Read targetToTotalRatio, relativeExpression. The explanation below defines the units, signs, ordering and any flags or placeholder values.
If the tool reports an input issue, correct the values and run it again. Apply the stated assumptions when interpreting a successfully returned result.

Input field	Meaning	Value type; see rules below	Requirement
targetBandIntensity	Target band intensity	number	Required
laneTotalProteinIntensity	Lane total protein intensity	number	Required
referenceLaneRatio	Reference lane ratio	number	Required

Enter nonnegative target intensity, positive total-lane intensity and positive reference ratio. Return target-to-total ratio and reference-normalized expression.

targetRatio=targetIntensity/totalLaneIntensity; relative=targetRatio/referenceRatio

Units, indexing, boundary conditions and exceptional cases are defined in the calculation rules above.

Output field	Meaning	Value type
targetToTotalRatio	Target to total ratio	number
relativeExpression	Relative expression	number

json

{
  "input": {
    "targetBandIntensity": 80,
    "laneTotalProteinIntensity": 200,
    "referenceLaneRatio": 0.5
  },
  "output": {
    "targetToTotalRatio": 0.4,
    "relativeExpression": 0.8
  }
}

Limitations and assumptions

Image segmentation, saturation, loading linearity and background are not assessed. Supply corrected comparable intensities and a ratio rather than raw reference intensity.

Numeric inputs must be finite. Follow the calculation-specific rules for overflow, underflow and exceptional cases. Missing required fields, extra fields, wrong value types and violations of the calculation rules are input errors.

Alternative or Complementary methods

Use Western Band Background Correction: Subtract local western-blot band background before lane normalization. For a complementary task, use BCA Blank Correction: Subtract the matched assay blank from BCA absorbance.

References

Western Total Protein Normalization — tool.io

Similar or alternative tools

Western Band Background Correction
Subtract local western-blot band background before lane normalization.
BCA Blank Correction
Subtract the matched assay blank from BCA absorbance.