Troubleshooting Citrate Synthase Protocol

Goal

• To create a protocol to quantify citrate synthase activities in Astrangia poculata

Samples

• Sampled extended polyps with scissors and foreceps
• Sampled one symbiotic (#1) and aposymbiotic (#2) coral

Overall workflow

1. Collect/thaw samples
2. Prepare and lyse samples
3. Quantify protein
4. Prepare samples
5. Add reagents to samples and +/- controls
6. Read on spectrophotometer (baseline)
7. Add Oxaloacetic Acid and read on spectrophotometer
8. Calculate

Preparation

This protocol is modified from Sigma-Aldrich Citrate Synthase Protocol, Hawkins et al. 2016, and Spinazzi et al. 2012.

Kits

• Sigma-Aldrich Citrate Synthase Kit
• Pierce BCA Protein Assay Kit

Equipment

• Vortex
• 96 well plate (flat bottom)
• Plate spectrophotometer
• Tabletop and larger centrifuges for 1.5mL tubes

Reagents/Supplies not included in kits

• DI water
• 100% ethanol
• Lysis Buffer
• ZR BBashing lysis tubes with 0.1mm and 0.5mm beads

Reagent Preparation

• Lysis buffer (25 mM Tris (pH 7.8), 1 mM EDTA, 10% gylcerol [v/v])
  • To make 100 mL of lysis buffer, add:
    • 2.5 mL 1 M Tris (pH 8)
    • 0.2 mL 0.5 M EDTA
    • 10 mL 100% glycerol [v/v]
    • 87.3 mL DI water
  • Store at room temperature
• Assay buffer (1X)
  • Solution comes as 5X in the kit, we need to dilute to 1X
  • Today I needed ~3.5 mL of assay buffer (for reaction, Acetyl CoA solution, and citrate synthase dilution)
    • 0.7 mL of 5X Assay buffer + 2.8 mL DI water = 3.5 mL 1X Assay buffer
• Acetyl CoA solution (30 mM)
  • Dissolve entire vial with 1 mL DI water
  • Mix until homogenous
  • Aliquot into 5 vials of 200 μL and store at -20°C
• Oxaloacetate (OAA) solution (10 mM)
  • Dissolve 1.3 mg of OAA powder in 1 mL of 1X Assay Buffer
  • Store at -20°C for maximum 1 week
• DNTB solution (10 mM)
  • Dissolve entire vial with 1 mL 100% Ethanol
  • Mix until homogenous
  • Aliquot into 5 vials of 200 μL and store at -20°C
• Citrate Synthase
  • Diluted 2 μL of Citrate Synthase solution with 60 μL of 1X Assay buffer

Protocol

1. Collect/Thaw samples

• Collected one polyp from a dark (symbiotic) and light (aposymbiotic) A. poculata colonies with scissors and foreceps
• Placed immediately into Bead tube with 500 μL of lysis Buffer

2. Prepare and lyse samples

• Vortex samples for ~2 minutes
• Remove liquid and transfer into a new, labelled 1.5 mL tube
• Centrifuge for 1.5 minutes at 16000 rcf
• Transfer supernatant to a new, labelled 1.5 mL tube, discard pellet
• Aliquot 80 μL for protein quantification and freeze the rest at -80 °C

3. Quantify protein

• I used the protocol from the Pierce BCA Protein Assay Kit
• My analysis for protein content (mg/mL) per sample can be found here
• Protein content in each sample
  • Sample 1: 0.774 mg/mL
  • Sample 2: 0.275 mg/mL

4. Prepare samples

• According to Hawkins et al. 2016, we need to dilute to a yield of 2-6 μg of protein in 20 μL of sample per well
  • Sample 1:
    • 0.774 mg/mL = 0.774 μg/μL
    • 0.774 μg/μL x 20 μL = 15.4 μg of Protein
    • Need to dilute by 3X
      • 6.6 μL of Sample 1 + 13.5 DI water = 20 μL with ~5.1 μg of protein
  • Sample 2:
    • 0.275 mg/mL = 0.275 μg/μL
    • 0.275 μg/μL x 20 μL = 5.2 μg of Protein
    • No need to dilute
• The above calculations are for 1 well, we need to multiply everything by 3 to have 3 replicate wells per sample
  • In 1.5 mL eppindorf tubes:
    • Sample 1: 19.8 μL Sample 1 + 40.2 DI water
    • Sample 2: 60 μL Sample 2
  • Store on ice
• For the negative control, add 60 μL of lysis buffer to a new, labeled 1.5 mL tube
• For the positive control, use the diluted citrate synthase tube from the reagent preparation step

5. Add reagents to samples and +/- controls

• Prepare a master mix of the reagents prior to adding it to the sample tubes
  • In each well, we need 170 μL of master mix with the following concentrations:
  • 3.3 μL of 30 mM Acetyl CoA solution (making it 588 μM)
  • 5 μL of 10 mM DNTB solution (making it 294 μM)
  • 161.7 μL of 1X Assay buffer
  • We have 2 samples, 1 negative control (lysis buffer) and 1 positive control (citrate synthase), with three replicates for each
    • Therefore we need enough master mix for 12 wells
    • I made enough for 15 wells for a total of 2550 μL of master mix
      • 49.5 μL of 30 mM Acetyl CoA solution (making it 588 μM)
      • 75 μL of 10 mM DNTB solution (making it 294 μM)
      • 2425.5 μL of 1X Assay buffer
• Mix well by vortexing
• Add 510 μL of master mix (170 μL x 3 replicates) to the sample and control tubes
• Mix well by vortexing
• Add 190 μL of sample into the corresponding wells on the 96 well plate

6. Read on spectrophotometer (baseline activity)

• On kinetic mode, I read these samples at 412 nm for 3 mins with a 49 second interval, resulting in 4 data points

7. Add Oxaloacetic Acid and read on spectrophotometer

• Add 10 μL of OAA solution to each well
• Use a multi-channel pipette to start all reactions simultaneously
• Mix well by pipetting
• Re-read the plate on the same settings as Step 6

8. Calculate

• I calculated the citrate synthase activity using RStudio and my script can be found here

Conclusions

• This protocol worked for A. poculata, with citrate synthase activities of:
  • Sample 1 (symbiotic) = 68.86 mU/mg
  • Sample 2 (aposymbiotic) = 101.51 mU/mg
• These values fall in the same range of citrate synthase activities in symbiotic Exaiptasia pallida sea anemones (Hawkins et al. 2016)
• However, our results differ from Hawkins et al. 2016, with our aposymbiotic colonies resulting in higher citrate synthase activities than the symbiotic colonies
  • After this preliminary test, we will test again with larger samples sizes to see if this trend holds true