Internal reference protein Generally refers to proteins encoded and expressed by housekeeping genes, which have relatively constant expression levels in various tissues and cells.

The role of reference proteins It is mainly used to correct errors in protein sample quantification and loading process, improve the accuracy of experimental results, and serve as a control for WB experiments.

Selection of internal reference proteins

①Based on the species source of the experimental samples, GAPDH, β - actin, β - tubulin, Lamin B, and Histone H3 are usually selected for mammalian samples, while actin and Rubisco are commonly used for plant samples.

②If detecting subcellular localization proteins, internal reference proteins corresponding to organelle localization can be selected, which can better reflect the accuracy of internal reference.

③Internal reference proteins generally require a molecular weight difference of at least 5KD from the target protein

Precautions for selecting internal reference proteins：

In glucose metabolism and hypoxia models, the expression of GAPDH in specific cells and tissues is increased, so GAPDH cannot be used as an internal reference; Cytoskeleton related research and skeletal muscle samples cannot use β - actin as an internal reference, as the expression level of β - actin is relatively low in adipose tissue and cells; Lamin B cannot be used as an internal reference in cytotoxicity experiments.

Histone H3 is expressed quite stably in the nucleus, and the hypoxia model can generally be modeled using Tublin. When comparing the expression levels of multi tissue and multi cell samples, it is best to choose GAPDH as an internal reference. GAPDH belongs to metabolic proteins and its expression is relatively constant in living tissues. When detecting phosphorylated proteins or induced samples, structural proteins such as β - actin or β - tubulin are usually chosen.

Reference Table for Internal Reference Proteins