If your instrument can capture only near-infrared signals, use Revert 700 Total Protein Stain, which can be visualized in the 700 nm channel. Membrane staining can verify that sample protein was uniformly loaded across the gel, and assess the quality and consistency of protein transfer. After transfer and prior to immunodetection, the membrane is treated with this fluorescent protein stain and imaged. Revert 700 Total Protein Stain is used to assess sample protein loading in each lane as an internal loading control. Revert™ 700 Total Protein Stain Kit ( /revertkit) Protein concentration must be determined for all samples. This protocol is intended for relative comparison of pan-protein and phospho-protein signals, and results do not indicate the stoichiometry of phosphorylation (1). Important guidelines are provided in Section Verify specificity of the phospho-antibody to ensure that it does not cross-react with the unmodified target protein, and to identify possible interference from background bands. The Antibody Publication Database can help you find antibody pairs that work for your experiment ( /antibodyrequest).Īntibody validation. Always perform single-color control blots first to verify antibody specificity, and to identify possible interference from background bands. 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. Housekeeping Protein Normalization Protocol Introduction
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