Secretory Granules: Methods for Preparation

Abstract

Secretory granules – small membrane‐bound compartments for transport of secretory proteins to the extracellular environment – regulate the secretion of peptide hormones such as insulin by mammalian cells. The granules need to be isolated and purified for studies of their formation and maturation. They can be purified by high‐speed gradient centrifugation or by immunoadsorption. Although immunoadsorption is faster and gives higher purity, it requires the identity of the surface antigens to be known.

Keywords: secretory granules; SNARE; ultracentrifugation; Golgi; immunoisolation

Figure 1.

Preparation of secretory granules by ultracentrifugation. Immature and mature secretory granules are isolated from cellular post‐nuclear supernatant (PNS) by a two‐stage ultracentrifugation procedure. Ultracentrifugation of the PNS through a velocity sucrose gradient isolates granules from other larger cellular membranes such as the Golgi complex. Further ultracentrifugation of granule‐enriched fractions on an equilibrium density gradient enables separation of immature and mature secretory granules based on their differences in density.

Figure 2.

Isolation and characterization of secretory granules by immunoadsorption. (a) Granules radiolabelled at different stages of maturation from pulse‐chased cells can be isolated by immunoadsorption with antibodies against a known granule surface antigen such as VAMP4. (b) The precise sorting pattern of a granule surface antigen during granule biogenesis can be determined using this method. Here the sorting of FLAG‐VAMP4 away from secretory granules during maturation is evident as the amount of radiolabelled POMC products recovered on anti‐FLAG beads decreased with increasing chase time. (c) The effects of drugs on granule maturation can also be analysed. Following Brefeldin A treatment, the prevalence of VAMP4 on mature secretory granules indicates that its sorting from granule membrane was blocked by this drug.

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References

Chavez RA, Chen YT, Schmidt WK, Carnell L and Moore HP (1994) Expression of exogenous proteins in cells with regulated secretory pathways. Methods in Cell Biology 43A: 263–288. [Review.]

Eaton BA, Haugwitz M, Lau D and Moore HP (2000) Biogenesis of regulated exocytotic carriers in neuroendocrine cells. Journal of Neuroscience 20 (19): 7334–7344.

Fernandez CJ, Haugwitz M, Eaton B and Moore HP (1997) Distinct molecular events during secretory granule biogenesis revealed by sensitivities to brefeldin A. Molecular Biology of the Cell 8 (11): 2171–2185.

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Moore, Hsiao‐Ping H, Chan, Charles KF, and Eaton, Benjamin A(Mar 2004) Secretory Granules: Methods for Preparation. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0002595]