This method for extracting protein from ground coral samples is based on the Bradford assay for the colorimetric detection and quantification of total protein (Bradford, 1976) and is compared to a known standard dilution of bovine serum albumin (BSA). Pierce Inc. and Bio-Rad have developed the reagents and standards necessary for completing the extraction. There are five parts to quantifying total soluble protein in ground corals: 1) grind and sub-sample the coral and store at -80 °C until ready to extract, 2) solubilize protein via cell disruption [detergent lysis and freeze-thaw lysis], 3) separate the dissolved protein from tissue and skeletal particles, 4) quantify the protein concentration via Bradford microassay procedure, and 5) standardize the protein concentration to ashfree dry weight (AFDW). This method was originally developed by Rowan McLachlan with the assistance of Jamie Price and Kerri Dobson and with the guidance of Dr. Noah Weisleder and Andréa Grottoli at The Ohio State University. This protocol was written by Rowan McLachlan and reviewed by all co-authors. dx.doi.org/10.17504/protocols.io.bdc8i2zw
Extraction of Total Soluble Lipid from Ground Coral Samples
This method is adapted and updated from methods originally published in Grottoli et al. (2004) and is based on the original methods of Folch & Stanley (1957), and Bligh & Dyer (1959). There are five parts to extracting lipids from ground corals: 1) grind and sub-sample the coral and store at -80 °C until ready to extract, 2) freeze-dry the sample, 3) extract the lipids from the freeze-dried samples, 4) standardize the lipid concentration to ash-free dry weight (AFDW), and 5) resuspend the extracted lipid for long-term storage and possible later analysis of lipid classes or isotopes. The lipid extraction procedure must be conducted in a fume hood with the sash as low as possible with the researcher wearing protective eyewear, gloves, and lab coat at all times. Important considerations regarding lipid analysis were gained from reading Chapter 1.3 “Lipid extraction, storage, and sample handling” from the textbook Lipid Analysis by Christie (2003). This method was originally developed by Andréa Grottoli and refined by Rowan McLachlan (06-11-18) with the guidance of Dr. Agus Muñoz-Garcia at The Ohio State University. This protocol was written by Rowan McLachlan (03-12-2020). dx.doi.org/10.17504/protocols.io.bc4qiyvw
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