Multiplex Sequential Immunoprecipitation of Insulin Secretory Granule Proteins from Radiolabeled Pancreatic Islets
Pulse radiolabeling of cells with radioactive amino acids is a common method for tracking the biosynthesis of proteins. Specific proteins can then be immunoprecipitated and analyzed by electrophoresis and imaging techniques. This chapter presents a protoc
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Introduction Pulse radiolabeling of cells followed by immunoprecipitation is a means of looking at the biosynthesis of targeted proteins [1, 2]. In this method, a radiolabeled amino acid is usually added to the medium so this can be incorporated into nascent proteins as they are being synthesized. Then the newly synthesized proteins can be immunoprecipitated by direct or indirect means and analyzed at any time, thereby providing a means of tracking their fate inside the cell. Direct immunoprecipitations are carried out by covalently linking an antibody of interest to a solid matrix such as cyanogen bromide (CNBr)-activated Sepharose [3, 4]. In this approach, antibodies are coupled directly to the resin through primary amines. Indirect immunoprecipitations are carried out by non-covalently binding the antibody to an affinity-based matrix such as Protein A Sepharose. Then in both direct and indirect protocols, cell lysates can be incubated with the resulting immunoadsorbents for binding, elution, and subsequent analysis of the target proteins.
Paul C. Guest (ed.), Multiplex Biomarker Techniques: Methods and Applications, Methods in Molecular Biology, vol. 1546, DOI 10.1007/978-1-4939-6730-8_14, © Springer Science+Business Media LLC 2017
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This chapter describes a 20 min pulse labeling of pancreatic islets with 3%S-methionine and the sequential immunoprecipitation of the hormone insulin and the secretory granule accessory proteins chromogranin A, prohormone convertase (PC)1 and PC2, as described in previous studies [5–7]. Enzymological analyses have shown that production of mature insulin requires cleavage of proinsulin by PC1 and PC2 on the carboxy-terminal side of Arg31-Arg32 and Lys64-Arg65, respectively [8]. PC1 has a pH optimum of 5.5 and requires mM calcion ion concentration, which coincides with the environment inside insulin secretory granules [2]. This fits with finding that final conversion of proinsulin to insulin does not begin to occur until approximately 30 min after initial synthesis on the rough endoplasmic reticulum (Fig. 1). Thus, the 20 min labeling period employed here helps to ensure that insulin is still present mostly in its precursor form, making Biosynthesis of insulin
Nucleus
Time 0 minutes
Rough endoplasmic reculum
Golgi complex 20 minutes
30 minutes
Trans Golgi network
Insulin secretory granules
60 minutes
Constuve secreon (not regulated)
Regulated secretion
Fig. 1 Diagram of a pancreatic islet cell showing the biosynthesis of insulin and transport through the regulated secretory pathway
Multiplex Analysis of Radiolabelled Islet Proteins
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quantitative studies more direct. Here, the preparation of immunoadsorbents, pulse radiolabeling of islets, immunoprecipitation, and gel-based analyses is presented.
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Materials 1. Purified monoclonal antibody for insulin (see Note 1). 2. Polyconal antisera for chromogranin A, PC1 and PC2 (see Note 2). 3. CNBr-Activated Sepharose 4 (GE Healthcare; Little Chalfont, Bucks, UK). 4. Activation solution: 1 mM
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