Protein Extractions from Amphistegina lobifera: Protocol Development and Optimization.

Michele Betti,Fabrizio Frontalini,Caterina Ciacci,Sigal Abramovich

doi:10.3390/life11050418

Michele Betti, Fabrizio Frontalini + Show 2 more

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https://doi.org/10.3390/life11050418

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Abstract

Proteins are essential to life, and the evaluation of their content, identification, and modification represents a fundamental assay in biochemistry research. Different analytical techniques and protocols have been specifically designed but have rarely been compared. Here, we test and compare a variety of methodologies and treatments for the quantification of proteins in Amphistegina lessonii, a larger symbiont-bearing benthic foraminiferal species. These analyses specifically include (a) lysis buffer (homemade vs. RIPA), (b) protein assays (Lowry, BCA, and Bradford), (c) ultrasonic bath treatment, and (d) protein staining (silver staining vs. Coomassie blue). On the basis of the comparative outcome, we suggest using the homemade lysis buffer, Lowry or BCA assays, ultrasonic bath treatment, and silver stain to maximize the extraction and characterization of protein for A. lessonii. This protocol might be suitable and extended to other benthic foraminiferal species, including the smaller ones.

Highlights

Proteins are essential to life, and the evaluation of their content, identification, and modification represents a fundamental assay in biochemistry research
Proteins are essential to life because they are functionally involved in a vast array of cellular activities from energy production to metabolism through DNA replication
The absorbance wasofmeasured at 700albumin (Lowry), 562 standards (BCA), and using a series bovine serum (BSA)

Summary

Introduction

Proteins are essential to life, and the evaluation of their content, identification, and modification represents a fundamental assay in biochemistry research. We test and compare a variety of methodologies and treatments for the quantification of proteins in Amphistegina lessonii, a larger symbiont-bearing benthic foraminiferal species These analyses include (a) lysis buffer (homemade vs RIPA), (b) protein assays (Lowry, BCA, and Bradford), (c) ultrasonic bath treatment, and (d) protein staining (silver staining vs Coomassie blue). On the basis of the comparative outcome, we suggest using the homemade lysis buffer, Lowry or BCA assays, ultrasonic bath treatment, and silver stain to maximize the extraction and characterization of protein for A. lessonii This protocol might be suitable and extended to other benthic foraminiferal species, including the smaller ones. The evaluation of protein content, its identification, and modification represent a fundamental assay in biochemistry researches that occasionally remains challenging [3] In this context, several analytical techniques and protocols designed for purification, protein extraction, quantification, and identification have been suggested [4]. Assay choice is carefully considered and is mainly related to the targeted biological samples, sample volume, recovery, protein aggregation, and chemical reactions [4], as well as the objective and applications

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