6/2/2023 0 Comments Western blot quantificationHow do different detection methods compare?Ĭolorimetry-based methods are well suited for qualitative analysis-determining the presence or absence of a protein-particularly from purified samples. Accurate quantitation relies on a sufficient SNR. The majority of the bands are still detectable, and allow precise quantitation even after 3 h.Īntibody specificity, membrane material, blocking agent, and detection/imaging system influence the ability to define signal from background. Amersham ECL Prime signal duration was monitored for 3 h. With a stable signal detection reagent, high sensitivity is maintained for, allowing multiple exposures and the possibility to detect weak bands that might be missed in a single, brief exposure.įig 3. Signal stability over time enables repeat exposures and the comparison of gels and membranes prepared at different times (Fig 3). The linear dynamic range is 3.6 orders of magnitude and very linear with a limit of detection of 1.2 pg. Dilution series of transferrin detected with C圓-labeled secondary antibodies. Measuring the linear dynamic range of a CCD camera-based imager.įluorescence detection with Amersham ECL Plex gives a broad linear dynamic range, making it possible to detect and compare weak and strong bands on the same blot (Fig 2).įig 2. A linear dynamic range that extends from high to low signal intensities supports accurate quantitation across an equally broad range of protein concentrations (Fig 1).įig 1. High sensitivity provides the ability to detect low-abundance proteins and influences the linear dynamic range. Signal-to-noise ratio (SNR): Clarity of signal over any nonspecific background.Signal stability: The ability to detect signal over time.Linear dynamic range: The signal intensity range that is equivalent to protein concentration.Sensitivity: Influences the lower limit of detection for low abundance proteins.In selecting a detection method, considerations include: An appropriate detection method affords the level of quantitation required by the experiment. The importance of quantitation will vary depending on the specific Western blot experiment. What factors influence the reliability of densitometry? The software can compare the measured density to a background area, typically adjacent to the target band, to produce a relative measurement. This area might be a specific protein band. Software algorithms determine the density of signal across a selected area. Detection methods include colorimetry and immunoblotting (via chemiluminescence, fluorescence, and radiolabeling). In Western blotting, densitometry quantitates proteins within the linear dynamic range of a chosen detection method. The technique uses standalone densitometers, imaging systems, or separate software. What is densitometry?ĭensitometry determines the optical density of a sample in solution, in-gel, or at a stage following transfer to membrane. Quantitative measurement relies on having a detection method with a known relationship between signal intensity and protein concentration across a useful range.Īn appropriate detection method is one that supports densitometry-a quantitative method-and provides sufficient accuracy to meet experimental goals. Western blot data are generally semiquantitative, and analysis often provides relative abundance, not absolute concentration.
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