Total Antioxidant Capacity Porites June Patch Adult tissues

The goal of this protocol is to determine the total antioxidant capacity (TAC) in adult coral tissues from the Porites Nutition June experiment using the OxiSelect Total Antioxidant Capacity Assay kit.

The protocol for this assay can be found here

Sample Description and Preparation

  • The samples used in this assay are from the airbrushed homogenates from this notebook entry
  • 500ul of homogenate were used per sample
  • Frozen homogenates were thawed on ice
  • To separate host and symbiont fractions, the thawed homogenates were centrifuged at 5200 RPM for 5 minutes at 4°C
  • The supernatant was transferred into two replicate tubes on ice:
    1. 100uL for TAC analysis
    2. 400uL for total protein analysis

Reagent preparation

Prepare all reagents and store at 4°C for a maximum of 3 months

1X Reaction Buffer

  • dilute to 1:100
  • 400uL kit Reaction buffer + 39.6mL of 1X PBS

1X Copper Ion Solution

  • dilute to 1:100
  • 1mL kit Reaction buffer + 99mL of DI H2O

1X Stop Solution

  • dilute to 1:10
  • 1.5mL kit Stop Solution + 13.5mL of DI H2O

Standard Preparation

  1. Prepare a 10 mg/mL (60 mM) uric acid solution with 1M NaOH
    • 10mg Uric Acid Powder + 1 mL 1M NaOH
  2. Dilute to a 2mM solution of Uric Acid
    • 100uL of 60mM Uric Acid + 2.9mL of DI H2O
  3. Create a serial dilution to match the table below:

Standard

  • For this entry, I did not use standards #4 and #7 to fit all of the samples on one plate

Assay Protocol

  1. Add 20uL of the Diluted uric acid standards or samples to the 96-well microtiter plate.
  2. Add 180uL of the 1X Reaction Buffer to each well using a multi-channel pipette. Pipette mix.
  3. Obtain initial absorbance by reading the plate at 490nm
  4. Add 50uL of the 1X Copper Ion Reagent into each well. Incubate for 5 minutes at room temperature on an orbital shaker.
  5. Add 50uL of the 1X stop solution to each well using a multi-channel pipette.
  6. Read plate again at 490nm

Plate layout

  1 2 3 4 5 6 7 8 9 10 11 12
A S1 S1 S2 S2 S3 S3 S5 S5 S6 S6 S8 S8
B S9 S9 S10 S10 489 489 501 501 513 513 525 525
C 537 537 549 549 561 561 573 573 585 585 597 597
D 609 609 621 621 633 633 645 645 657 657 669 669
E 681 681 693 693 705 705 717 717 729 729 741 741
F 753 753 765 765 777 777 789 789 801 801 813 813
G 825 825 837 837 849 849 861 861 873 873 885 885
H 897 897 909 909 921 921 933 933 945 945 957 957
Written on October 4, 2019