Two-color Western blot detection requires careful selection of primary and secondary antibodies to prevent cross-reactivity. ![]() However, these reagents do not preclude the need to perform a protein concentration assay before sample preparation and loading.Īntibody validation. You can use reagents designed to confirm uniform sample loading, such as Odyssey Loading Indicators (P/N 926-20002), to improve the accuracy of this validation protocol. A protein concentration assay (BCA, Bradford, or similar assay) must be used to adjust sample concentration and load all samples as consistently as possible. Uniform loading of total sample protein across the gel is critical for accurate QWB analysis. Replicates are discussed further on page Normalization Calculations and Analysis of Replicates. A minimum of three technical replicates is recommended for each sample. Replicate samples provide information about the inherent variability of your methods, to determine if the changes you see are meaningful and significant. See the protocol: Determining the Linear Range for Quantitative Western Blot Detection ( /LinearRange) for more information. Use a dilution series to verify that you are working within the linear range of detection, and signal intensity is proportional to sample loading. Saturated bands and sample overloading frequently compromise the accuracy of QWB. This protocol is intended for use with near-infrared fluorescent Western blots. This protocol describes how to use a housekeeping protein for Western blot normalization and quantitative analysis. For more information, see the Housekeeping Protein Validation Protocol ( /HKP-Validation LI-COR). However, expression of common HKPs is now known to vary in response to certain experimental conditions, including cell confluence, disease state, drug treatment, and cell or tissue type.īefore an HKP is used for Western blot normalization, stable expression must be validated for the specific experimental context and treatments. Because HKP normalization relies on a single indicator of sample loading, variation in HKP expression leads to inconsistent estimation of sample loading and introduces experimental error that may alter data analysis.įor widely-used HKPs (such as actin, tubulin, and GAPDH), stable expression has generally been assumed. Accurate normalization requires stable expression of the HKP across all experimental conditions and treatments. Housekeeping proteins (HKPs) are routinely used as loading controls for Western blot normalization. Using a Housekeeping Protein (HKP) as an Internal Loading Control The internal loading control is used as an indicator of sample protein loading, to correct for loading variation and confirm that observed changes represent actual differences between samples.įor more normalization related resources, see " Further Reading". 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. Since the area intensity is in arbitrary unit, it can also be normalised to the BCA assay measurement, DNA content or any other number chosen.Housekeeping Protein Normalization Protocol Introduction ![]() To normalise the intensity of the area underneath the peak to the Ponceau staining, measure the intensity of 3 randomly chosen peaks on the Ponceau image, average the measurements and use that value to normalise the data against. The report will automatically pop up on the side. Go to: Analyse→Gels→Label Peaks to get the report.Īlternatively, use the magic wand tool to highlight the area underneath the peak for each lane. Draw the line at where the peak begins and ends (bend in the line) for each peak. Use the line tool to draw the lines to eliminate the lane background from the calculations. Continue this for the subsequent lanes (pressing Crtl and 2 every time).įor the last lane, repeat the procedure but press Ctrl and 3 to set the last lane. Press Ctrl and 1 to set first lane (Command and 1 on the Mac).Ĭlick the centre of the square and drag it across to the next lane. Use the square selection tool to highlight the first lane. Convert the image to 8-bit using ImageJ function (Image→Type→8-bit).
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