Goals

1. Test the correct pH for total protein extractions. It should be at ~ pH 7 after neutralizing with 0.1 M HCl.

2. Test total protein concentrations from the holobiont, coral, and symbiont fractions.

GOAL 1: pH testing

In vials with 200uL of coral homogenate, I added 10uL of 1M NaOH with different volumes of 0.1 M HCl: 0uL, 50uL, 75uL, and 80uL.

From this test, it looked like 80uL was adequate to neutralize the sample. Then I tested the pH of one sample after the 10ul 1M NaOH addition, 4 hour incubation at 50C, and 80uL of 0.1M HCl. (Pictured below).

Result: 80uL of 0.1M HCl is enough to neutralize 1M NaOH. This volume was used for Goal 2.

GOAL 2: holobiont, coral, and symbiont fractions

Sample preparation

Holobiont Fraction:

• Thaw homogenate from -80C freezer
• Vortex sample
• Transfer 200uL of homogenate into corresponding centrifuge tube.

Coral Fraction:

• Transfer 100uL of homogenate into a new centrifuge tube.
• Centrifuge for 3 minutes at 15000 rcf
• Remove 200uL of the supernatant and transfer to the “Coral Fraction” tube
• Save the rest of the supernatant and store at -80C

Symbiont Fraction:

• Re-suspend pellet in 1000uL of 1X PBS (Phosphate Buffer Saline)
• Pipette mix and vortex until homogenized. Spin down.
• Remove 200uL of the symbiont fraction to the “Symbiont Fraction” tube
• Save the rest of the symbiont fraction and store at -80C

Vial	Fragment.ID	Timepoint	Fraction
1	R26	A2	Holobiont
2	R26	A2	Coral
3	R26	A2	Symbiont
4	R8	A2	Holobiont
5	R8	A2	Coral
6	R8	A2	Symbiont
7	R7	A1	Holobiont
8	R7	A1	Coral
9	R7	A1	Symbiont

Results

• The values correspond with other holobiont or coral fractions of Porites spp.