Background

• Analyzing citrate synthase concentrations from host and symbiont fractions (from the homogenate prep protocol) for the Astrangia nutrition project
• After separating each fraction, protein concentrations for all host and symbiont fractions were quantified using the BCA Protein Assay
• Only samples with >1 μg/20 μL of protein were used (the suggested range of protein is between 1-6 μg/20 μL)
• Samples with >6 μg/ 20 μL were diluted in half
• The citrate synthase protocol was used, with volume modifications to accommodate 150 wells

Samples

• The samples used in this experiment were:
  • 15-H, 21-H, 27-H, 33-H, 29-H, 45-H, 51-H, 57-H, 69-H, 75-H, 81-H, 87-H, 93-H, 99-H, 105-H 111-H, 117-H, 123-H, 129-H, 135-H, 147-H, 153-H, 21-S, 33-S, 39-S, 45-S, 51-S, 75-S, 81-S, 87-S, 105-S, 111-S, 117-S, 123-S, 129-S, 135-S, 153-S, 159-H, 165-H, 171-H

Master Mix

• Calculations
  • (73 samples + 2 standards) x 2 replicates = 150 wells
• For 150 wells:
  • 3.3 μL x 150 = 495 μL Acetyl CoA
  • 5 μL x 150 = 750 μL DNTB
  • 161.7 μL x 150 = 24.255 mL Assay Buffer

Assay Buffer (1x) Prep

• Need 24.255 mL + 1 mL for OAA = ~26 mL
  • 26 mL DI water + 5.2 mL Assay Buffer (5x)

Analysis

• The analysis and plate layout can be found in this GitHub repository