SAM ELISA Kit for A. poculata nutrition experiment

This protocol is to quantify SAM (S-Adenosylmethionine) quantities in Astrangia poculata. I used the SAM ELISA KIT from BioVision. A modified version of the kit protocol was used. Only 1-96 well plate is provided with this kit, therefore only 40 samples can be measured with 8 standards and duplicates. The following table shows the 39 samples selected for this analyses (based off a range of coloration in each treatment):

Table 1. Plug numbers and sample sizes from each tank and treatment.

Treatment Tank # of Samples Plug #s
No Light 1 7 2012, 1765, 1411, 2304, 2067, 1679, 1709
No Light 6 6 2564, 2517, 2735, 2858, 2407, 2014
Light 2 7 1606, 1329, 1695, 1436, 2015, 1051, 1811
Light 4 6 1264, 1534, 1235, 2164, 1260, 2863
Light Feed 3 7 2525, 1275, 1316, 1756, 1246, 2753, 1353
Light Feed 5 6 2882, 2513, 2070, 2410, 2189, 2184

Table 2. The corresponding vial number of coral homogenate to the plug number:

Plug # Vial #
2012 119
1765 167
1411 161
2304 227
2067 71
1679 185
1709 23
2564 59
2517 281
2735 275
2858 149
2407 233
2014 47
1606 251
1329 125
1695 77
1436 215
2015 209
1051 65
1811 29
1264 83
1534 191
1235 101
2164 155
1260 257
2863 197
2525 137
1275 35
1316 131
1756 41
1246 95
2753 17
1353 239
2882 245
2513 113
2070 203
2410 269
2189 53
2184 173

Equipment and Reagents needed

Reagent Preparation

  • Biotin detection antibody working solution
    • 96 wells x 0.05 mL = 4.8 mL –> 5 mL
    • Dilute Biotin Antibody with Antibody-dilution buffer at 1:100
    • 0.05 mL Biotin Antibody + 4.95 mL Antibody-dilution buffer
  • HRP-Streptavidin Conjugate (SABC) working solution
    • 96 wells x 0.1 mL = 9.6 mL –> 10 mL
    • Dilute SABC with SABC dilution buffer at 1:100
    • 0.1 mL SABC + 9.9 mL SABC dilution buffer
  • Wash Buffer
    • Dilute 30 mL of Wash buffer in 720 mL of DI water for a total of 750 mL
    • Incubate at 40°C to dissolve any remaining crystals
    • Cool to room temperature before use
  • Standard Preparation
    • Reconstitute lyophilized SAM standard by adding 1 mL of Standard/Sample dilution buffer to the vial. This concentration will be 25 ug/mL. Use within 2 hours of reconstituting
    • Allow solution to sit at room temperature of for 10 minutes, the gently vortex
    • Perform a series of serial dilutions to create the following range of standard concentrations in Table 3.
      • e.g. To prepare 0.6 mL of ug/mL standard (S2), add 0.3 of the stock solution (S1) with 0.3 mL of Standard/Sample dilution buffer.
      • S8 will be 300 uL of Standard/Sample dilution buffer

Table 3. Standard Concentrations

Vial ID Concentration ug/mL
S1 25
S2 12.5
S3 6.25
S4 3.125
S5 1.56
S6 0.781
S7 0.39
S8 0

Sample Preparation

  1. Thaw homogenate samples from -80°C
  2. Separate host from symbionts
    • Centrifuge samples at 6000g for 2 mins at 4°C
    • Remove 400 uL of supernatant and transfer to a new tube
  3. Add protease inhibitor cocktail
    • Since samples at 400 uL, 4 uL of protease inhibitor cocktail was added
    • Vortex to mix
  4. Lyse cells with multiple freeze thaw cycles
    • Place samples in -80°C for 5 minutes, the move directly into the thermocycler for at 37°C for 5 minutes. Repeat 5 times.
  5. Centrifuge to remove debris
    • Centrifuge at 5000g for 5 minutes

Assay Protocol

  1. Bring all reagents, standards, and samples to room temperature for 30 minutes
  2. Wash plate 2 times with 1 X Wash Solution before adding standard, sample, and control wells
  3. Add 50 uL of each standard or sample into appropriate wells
    • Immediately add 50 uL of Biotin-detection antibody working solution to each well
    • Cover with the Plate sealer provided
    • Gently tap to ensure wells are mixed
    • Incubate for 45 minutes at 37°C
  4. Discard the solution and wash 3 times with 1 X Wash Solution.
    • Wash by filling each well with 350 uL of Wash Buffer using a multichannel pipette. Let it soak for 1-2 minutes, and then remove all residual wash-liquid from the wells by aspiration.
    • After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Clap the plate on absorbent filter papers or other absorbent materials.
  5. Add 100 uL of SABC working solution into each well. cover the plate and incubate at 37°C for 30 minutes.
  6. Discard the solution and wash 5 times with 1 X Wash Solution as Step 4.
  7. Add 90 uL of TMB substrate into each well, cover the plate and incubate at 37°C in the dark for 15-30 minutes. The shades of blue should be seen in the first 3-4 wells by the end of the incubation.
  8. add 50 uL of the stop solution to each well. Read result at 450 nm within 20 minutes.

Calculations

The calculations for to determine the concentration of SAM can be found in this script.

Written on May 15, 2019