The high variability of faint bands introduces error that may affect data analysis and interpretation. They are difficult to discriminate from membrane background and may not be statistically significant. Faint bands that are near the lower limit of detection generally have much larger coefficients of variation. If the HKP is not detected in the linear range of sample loading, these strong bands will be underestimated and normalization will not be accurate. With two proteins of very different abundance, such as a target protein and highly expressed HKP loading control, it is absolutely critical to detect both proteins within the linear range. Compare the linear ranges to determine the amount of sample you should load to produce a linear response for both target and control. This range must be determined individually for the target protein and internal loading control. This protocol is intended for use with near-infrared fluorescent Western blots. This protocol explains how to use serial dilutions of sample protein to determine the linear ranges of detection for a target and internal loading control, and choose an appropriate amount of sample to load for QWB analysis. Increasing amounts of target do not produce a proportional increase in the recorded signal. Signal saturation occurs when the signal intensity of a band is too bright for the detection system to record.Highly abundant proteins may also exceed the local binding capacity of the membrane and be washed away. When samples are overloaded, abundant proteins can bind in layers on the membrane surface that limit antibody access during detection. It frequently interferes with accurate detection of abundant proteins, including HKP loading controls. Membrane saturation is the result of sample overloading.Error introduced by saturation may alter data analysis and interpretation. Intensity of strong bands is underestimated, interfering with comparison of relative protein levels across the blot. Saturated bands yield similar intensity values, regardless of the actual amount of target present, and cannot be accurately quantified. Saturation occurs when increasing amounts of target fail to produce the expected increase in band intensity. Dotted lines indicate loss of proportionality on the upper and lower ends of the linear range. Combine these ranges to identify a level of sample loading that will produce a linear response for both. Determine the linear range separately for the target and internal loading control. In the linear range of detection, the target and internal loading control both display a linear relationship between sample loading and band intensity. Band intensity no longer reflects the abundance of target, and quantification is not possible. At the upper and lower ends of the linear range, this proportional relationship is lost. For example, a two-fold increase in sample loading is expected to produce a two-fold increase in band intensity. A change in target abundance should produce an equivalent change in signal response. Within the linear range of detection, band intensity should be proportional to the amount of target. Outside this range, signal intensity is not dependent on sample loading and does not accurately reflect the amount of target. In QWB analysis, the linear range of detection is the range of sample loading that produces a linear relationship between the amount of target on the membrane and the band intensity recorded by the detector ( Figure 45). Linear Range, Saturation, and Proportional Signals The combined linear range is then used to determine how much sample should be loaded to produce a linear signal response for both the target protein and the internal loading control ( Figure 45).Įmpiria Studio® Software provides a dedicated workflow for this process. QWB analysis is accurate only if the target protein and internal loading control can both be detected within the same linear range – a range that must be determined experimentally for each target and loading control. ![]() The internal loading control is used as an indicator of sample protein loading, to correct for loading variation and confirm that changes observed in target protein bands represent actual differences between samples. In quantitative Western blotting (QWB), normalization mathematically corrects for unavoidable sample-to-sample and lane-to-lane variation by comparing the target protein to an internal loading control. Determining the Linear Range for Quantitative Western Blot Detection Introduction
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