Secretion of Adipsin as an Assay to Measure Flux from the Endoplasmic Reticulum (ER).

In this protocol we describe a quantitative biochemical assay to assess the efficiency of endoplasmic reticulum (ER) to Golgi protein transport in adipocytes (Bruno ., 2016). The assay takes advantage of the fact that adipocytes secrete various bioactive proteins, known as adipokines. As a measure of ER to Golgi flux we determine the rate of bulk secretion of the adipokine adipsin post washout of Brefeldin A (BFA) treatment using immunoblotting. Because BFA treatment results in an accumulation of adipsin in the ER, the exit of adipsin from the ER upon BFA washout is synchronized across cells and experimental conditions. Thus, using this simple assay one can robustly determine if perturbations, such as knocking down a protein, have an effect on ER to Golgi protein transport.