Secreted Proteins Undetectable in Cell Lysate (Western Blot (CST Guide))

Question

Accepted Answer

Target protein cannot be reliably detected in whole cell extract despite confirmed expression. Signal may appear in cell culture media but not in cell lysate. Common causes: 1) Target protein is secreted from cells and primarily exists in culture media rather than intracellular compartments 2) Insufficient concentration of secreted protein in media for detection 3) Active secretion machinery exports protein before lysis Solutions: 1) Collect and concentrate cell culture media using acetone precipitation or ultrafiltration concentrators 2) Use Brefeldin A (#9972) to inhibit protein secretion and retain target protein inside cells for detection in lysate 3) Compare cell lysate and concentrated media side-by-side: example shows cleaved caspase-1 more easily detectable in concentrated media than whole-cell extracts 4) Verify secretion using PhosphoSitePlus database and literature references for your target protein