Putnam Lab Total Protein Protocol

Draft Protocol

Goal: To quantify the total protein concentration in coral homogenate samples.

Written for quantifying total protein of Montipora capitata and Pocillopora acuta fragments from the Holobiont Integration 2018 project.

Based on the following Putnam Lab Airbrushing protocols:
K. Wong protocol

This protocol is adapted to determine total protein content using the Pierce BCA Protein Assay Kit from Thermo Scientific.

Summary Protocol

1. Prepare the tissue homogenate sample using NaOH, 4 hour incubation period, and HCl prior to total protein procedure.
2. Prepare BSA Standards.
3. Prepare the BCA working reagent.
4. Microplate procedure from Pierce BCA Protein Assay Kit.

Detailed Protocol

Adult Tissue Sample Preparation

1. Thaw homogenate aliquot labeled with the coral ID and “total protein”.
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) to the tube.
6. Pipette a very small amount of sample onto pH paper to confirm the pH ~10.
7. Incubate the tube at 50°C for 4 hours shaking at 300 rpm.
8. 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.

For coral larvae samples, see K. Wong protocol.

Preparation of Diluted Albumin (BSA) Standards
These standards can be made during the 4 hour incubation period in the sample preparation section.

1. 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.).

Vial	Volume of Diluent (μL)	Volume of Source of BSA (μL)	Final BSA Concentration (μg/mL)
A	0	300 of Stock	2000
B	125	375 of Stock	1500
C	325	325 of Stock	1000
D	175	175 of vial B dilution	750
E	325	325 of vial C dilution	500
F	325	325 of vial E dilution	250
G	325	325 of vial F dilution	125
H	400	100 of vial G dilution	25
I	400	0 (Blank)	0

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 + 10 samples) x (2 replicates) x (200 μL of WR) = 7,600 μL WR
   (9 standards + 20 samples) x (2 replicates) x (200 μL of WR) = 11,600 μL WR
   (9 standards + 40 samples) x (2 replicates) x (200 μL of WR) = 19,600 μL WR

2. Prepare WR by mixing 50 parts of BCA Reagent A with 1 part of BCA Reagent B (50:1, Reagent A:B) in a sterile glass bottle of the appropriate size based on how many samples are going to be run.

Microplate Procedure (Sample to WR ratio = 1:8) from Pierce BCA Protein Assay Kit:

1. Pipette 25 μL of each standard or unknown sample replicate into a microplate well.
2. Add 200 μL of the working reagent (WR) to each well and mix the plate thoroughly on a plate shaker for 30 seconds.
3. Cover the plate and incubate at 37°C for 30 minutes.
4. 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.
5. 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.