Housekeeping Protein (HKP) Validation Protocol v1

Housekeeping proteins (HKPs) are routinely used for Western blot normalization. For common HKPs (such as actin, tubulin, or GAPDH), stable protein expression is generally assumed. 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. Because HKP normalization uses a single indicator of sample loading, changes in HKP expression will introduce error and may alter data analysis and interpretation. Before 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. 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. Note: This protocol is intended for use with near-infrared fluorescent Western blots.