![]() ![]() ![]() 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 fluorescent Western blots. This protocol explains how to validate an HKP for use as an internal loading control, by demonstrating that HKP expression is stable in the relevant experimental samples. Because HKP normalization uses a single indicator of sample loading, changes in HKP expression or stability will introduce error and may alter data analysis and interpretation.īefore using a housekeeping protein for Western blot normalization, it is critical to validate that its expression is constant across all samples and unaffected by the specific experimental context and conditions. However, expression of several HKPs is now known to vary in response to certain experimental conditions, including cell confluence, disease state, drug treatment, and cell or tissue type. For common HKPs (such as actin, tubulin, or GAPDH), stable protein expression is generally assumed. Housekeeping proteins (HKPs) are routinely used for Western blot normalization. Using a Housekeeping Protein for Normalization 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 Validation Protocol Introduction ![]()
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