The Protein Journal

, Volume 37, Issue 4, pp 380–389 | Cite as

Biochemical Analyses of Proteins from Duttaphrynus melanostictus (Bufo melanostictus) Skin Secretion: Soluble Protein Retrieval from a Viscous Matrix by Ion-Exchange Batch Sample Preparation

  • Douglas O. C. Mariano
  • Marcela Di Giacomo Messias
  • José Pedro Prezotto-Neto
  • Patrick J. Spencer
  • Daniel C. Pimenta


A crucial step in scientific analysis can be sample preparation, and its importance increases in the same rate as the sensitivity of the following employed/desired analytical technique does. The need to analyze complex, viscous matrices is not new, and diverse approaches have been employed, with different success rates depending on the intended molecules. Solid-phase extraction, for example, has been successfully used in sample preparation for organic molecules and peptides. However, due to the usual methodological conditions, biologically active proteins are not successfully retrieved by this technique, resulting in a low rate of protein identification reported for the viscous amphibian skin secretion. Here we describe an ion-exchange batch processing sample preparation technique that allows viscous or adhesive materials (as some amphibian skin secretions) to be further processed by classical liquid chromatography approaches. According to our protocol, samples were allowed to equilibrate with a specific resin that was washed with appropriated buffers in order to yield the soluble protein fraction. In order to show the efficiency of our methodology, we have compared our results to classically prepared skin secretion, i.e., by means of filtration and centrifugation. After batch sample preparation, we were able to obtain reproductive resolved protein chromatographic profiles, as revealed by SDS-PAGE, and retrieve some biological activities, namely, hydrolases belonging to serine peptidase family. Not only that, but also the unbound fraction was rich in low molecular mass molecules, such as alkaloids and steroids, making this sample preparation technique also suitable for the enrichment of such molecules.


Batch chromatography Ion-exchange chromatography Viscous sample Amphibian skin secretion Hydrolase 



We are thankful to Dr. Ivo Lebrun for the thoughtful considerations about the batch sample preparation and for providing the IEX media.


This work was funded by CAPES (DOCM, 969130 Grant), FINEP (Financiadora de Estudos e Projetos) (Grant Number 01.09.0278.04), FAPESP and CNPq. DCP is a CNPq fellow researcher (Conselho Nacional de Desenvolvimento Científico e Tecnológico) (Grant 303792/2016-7).

Compliance with Ethical Standards

Conflict of interest

Authors declare that they have no conflict of interest.

Ethical Approval

This article does not contain any studies with animals performed by any of the authors.

Supplementary material

10930_2018_9780_MOESM1_ESM.docx (930 kb)
Supplementary material 1 (DOCX 930 KB)


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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Laboratório de Bioquímica e BiofísicaInstituto ButantanSão PauloBrazil
  2. 2.Centro de BiotecnologiaIPENSão PauloBrazil

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