Experiments

Below is a detailed review of our ELISA protocol for testing the binding between ferritin and our aptamer. Details on our computational docking protocols and tools have been added to the model page.

ELISA:

Experiment: Create an Enzyme-Linked Immunosorbent Assay (ELISA) to measure concentration of ferritin using the peptide ligand or DNA aptamer that we found through literature review and computational docking. Find out if peptide ligand/DNA aptamer successfully binds to ferritin. Find out whether peptide or DNA binds more successfully to ferritin.

ELISA Protocol Overview:

    1. Ferritin is added to 96 well plates at different concentrations.

    2. The aptamer (DNA or peptide), tagged with biotin, is added so that it can bind to ferritin.

    3. A small protein called Streptavidin conjugated with an HRP enzyme is added and the streptavidin binds to biotin.

    4. A substrate is then added to react with the HRP enzyme and produce a chemiluminescent signal proportional to the concentration of ferritin in the well.

    5. A plate reader then reads the absorbance of the signal produced by applying a 450nm wavelength, and the ferritin concentration is determined based on a standard curve of absorbance vs concentration generated from standard (known concentrations) ferritin samples.

elisa-experiment-steps

Visual of ELISA Steps Listed Above Made Using Biorender

Trial 1:

  • We ran two plates.
  • Used commercially available ferritin ELISA kit for reagents and standards (to create standard curve).
  • ELISA kit contained recombinant ferritin to use as standard and biotinylated rabbit anti-ferritin antibody
  • We tested the peptide on our samples in the ELISA (not the standards).
  • We used native ferritin for our samples because ferritin in human saliva is native and not recombinant.
  • Plate 1
    • Standards: Recombinant ferritin, antibody added.
    • Samples: Native ferritin, peptide added.
  • Plate 2:
    • Standards: Native ferritin, antibody added.
    • Samples: Native ferritin, peptide added.

Plate Map:

  • Ferritin added at concentrations in ng/mL shown below.
  • Blank = no ferritin added, control (should result in absorbance reading of 0).

elisa-trial-1-plate-map

Plate Map for Trial 1 (Stadard was recombinant ferritin + antibody for plate 1, native ferritin + antibody for plate 2)

Computational Docking:

We did not add much here on the computational docking done prior to ELISA here as we go into detail on the tools and protocols used on the model page.

DNA Shift Assay:

Experiment: Run a DNA shift assay experiment in order to determine if the DNA aptamer we found through literature review and computational docking successfully binds to ferritin. This experiment uses an agarose gel to visualize the migration pattern of DNA, ferritin, and DNA ferritin complexes. Smaller sized samples migrate further through the gel because they more easily fit through the pores of the gel. Larger sized samples do not migrate through the gel as far as smaller sized samples because they have a harder time moving through the pores of the gel. The samples in the gel are seen as bands under UV light.

DNA Shift Assay Protocol Overview:

    1. Tris EDTA (TE) buffer is prepared.

    2. 1.5% agarose solution in TE buffer is prepared, and ethidium bromide (binds to DNA and ferritin to allow for detection of bands in gel under UV light).

    3. The agarose was poured into a gel casting tray in order for the gel to set.

    4. Samples were prepared in microtubes.

      a. 6 Samples: Ferritin, DNA aptamer, and four different concentrations of ferritin with DNA aptamer added to allow for DNA to bind to ferritin.

      b. The goal was to see the difference in migration in the gel between these samples.

        i. DNA should migrate the furthest since it is the smallest.

        ii. If DNA successfully binds to ferritin, the complex should migrate through the gel more slowly compared to ferritin on its own so the complex will not migrate as far in the gel.

        iii. For the ferritin-DNA complex samples, there might also be two bands visible due to excess DNA that did not bind to ferritin.

    5. 1x non SDS loading dye was added to each sample to ensure that samples were successfully loaded into the wells of the gel and so that band migration could be visible when running the gel. SDS denatures proteins to allow for each band of the protein to be visible in the gel. We needed an intact native ferritin structure to allow for DNA binding so we did not use SDS.

    6. TE buffer was poured into the chamber so that it covered the gel.

    7. All six samples were loaded into the wells (six wells total loaded).

    8. The electrodes of the electrophoresis chamber were attached to a power source, and the gel was run at 100 Volts for an hour.

    9. The gel was then visualized under UV light.

dna-steps

Visual of DNA Shift Assay Steps Listed Above Made Using Biorender

chamber-photo

Photo Taken of Gel Electrophoresis Chamber Set Up

Trial 1:

  • We ran one gel.
  • We prepared and loaded six samples at 30uL per well
  • Lane 1: DNA (1ug)
  • Lane 2: Native Ferritin (40ug)
  • Lane 3: Native Ferritin (5ug) + DNA (20ug)
  • Lane 4: Native Ferritin (10ug) + DNA (20ug)
  • Lane 5: Native Ferritin (20ug) + DNA (20ug)
  • Lane 6: Native Ferritin (30ug) + DNA (20ug)