Protein Content of Madracis decactis and Diploria labyrinthiformis tissue for Physiological Analyses

This protocol was adapted from the proceedure provided by the Pierce BCA Protein Assay Kit from Thermo Scientific

Contents

• Materials
• Protocol
• Standard Table
• References

1. Materials
   • Pierce BCA Protein Assay Kit from Thermo Scientific
   • clear 96 Well plate
   • [Incubator[(https://www.scientificindustries.com/incubator-genie.html) or Waterbath with range from 37°C to 50°C.
   • Plate reader Spectrophotometer
   • 1M NaOH
   • 0.1M HCl
   • Pipettes P10, P200, P1000 and tips
   • 1.5ml microfuge tubes
   • DI water
2. Protocol

Adult Tissue Sample Preparation for Insoluble Protein from Holobiont

1. Thaw a 500 μL aliquot of tissue homogenate.
2. Vortex to re-suspend the symbiont cell pellet.
3. Label a new microcentrifuge tube with the coral ID, total protein assay date, and “prot”. If aliquot tube already contains exactly 500 μL of tissue sample, no need to make a new microcentrifuge tube.
4. Pipette 500 μL of the adult coral tissue sample into the new labeled 1.5 mL microcentrifuge tube.
5. Add 10 μL of 1M NaOH (pH should be ~10) in the tube.
6. Invert tube to mix the 1M NaOH in the tissue homogenate.
7. Pipette a very small amount of sample onto pH paper to confirm the pH ~10.
8. Incubate the tube at 50°C for 4 hours flicking to mix throughout to solublize protein with the use of the incubator at 35rmp
9. Add 280 μL of 0.1M HCl to the tube to neutralize the sample. Add this volume in small amounts and continue to test the pH of the sample using pH paper. pH needs to be at 7.0 to move onto the next steps.
10. Invert tube to mix
11. It is critical to record exactly how much volume of NaOH and HCl was added

Preparation of Diluted Albumin (BSA) Standards

1. Dilute the contents of one Albumin Standard (BSA) ampule into several clean vials, preferably using the same diluent as the samples. These standards can be made during the 4 hour incubation period in the sample preparation section.
2. Use the following table as a guide to prepare a set of protein standards. For this project we will use the microplate procedure. Diluent is DI water Type II. Each vial will be a sterile 1.5 mL microcentrifuge tube. Label the cap of the microcentrifuge tube with the Vial ID (“A”, “B”, etc.).

Standard Table

Preparation of the BCA Working Reagent (WR)

1. Use the following formula to determine the total volume of WR required:
   (# standards + # unknowns) x (# replicates) x (volume of WR per sample) = total volume WR required
   For this project, we will use 9 standards and 200 μL of WR is required for each sample in the microplate procedure.

   (9 standards + # samples) x (2 replicates) x (200 μL of WR) = total volume WR required
   (9 standards + 5 samples) x (2 replicates) x (200 μL of WR) = 5,600μL = 5.6mL WR (Prepare 7)

2. Prepare WR by mixing 50 parts BCA Reagent A with 1 part BCA Reagent B (50:1, Reagent A:B). For the above example, combine 10 mL of Reagent A with 0.2 mL of Reagent B.
   • Note: When Rapid Gold BCA Reagent B is first added to BCA Reagent A, a pale blue precipitate may be observed, but, upon vortexing or mixing for < 5 seconds, the precipitate should dissolve to yield a clear, green solution.
3. Microplate Procedure (Sample to WR ratio = 1:8) from Pierce BCA Protein Assay Kit:
   • Pipette 25 μL of each standard or unknown sample into microplate wells in duplicate.
   • Add 200 μL of the working reagent (WR) to each well and mix the plate thoroughly on a plate shaker for 30 seconds.
   • Cover the plate and incubate at 37°C for 30 minutes.
   • Cool plate to RT. Measure the absorbance at or near 562 nm on a plate reader Spectrophotometer. Wavelengths from 540–590 nm have been used successfully with this method. Spectrophotometer
   • Subtract the average 562 nm absorbance measurement of the Blank standard replicates from the 562 nm measurements of all other individual standard and unknown sample replicates.
   • Pepare a standard curve by plotting the average Blank-corrected 562nm measurement for each BSA standard vs. its concentration in μg/mL. Use the standard curve to determine the protein concentration of each unknown sample.