Results

ELISA Trial 1 Results:

Plate 1 Results:

elisa-trial-1-plate-1-results

ELISA (ferritin + peptide aptamer) Trial 1 Plate 1 (Standard was recombinant ferritin + antibody)

Plate 2 Results:

elisa-trial-1-plate-2-results

ELISA (ferritin + peptide aptamer) Trial 1 Plate 2 (Standard was native ferritin + antibody)

Notes:

  • Inconclusive results.
  • The commercial kit that was used did not come with a blocking buffer, and relied on a sample diluent with some blocking buffer on it. Blocking buffer is needed to prevent nonspecific binding of antibody/peptide, streptavidin-hrp, or substrate on the wells by blocking out empty spots on the wells that ferritin did not bind to. The sample diluent in the commercial kit was ineffective at blocking the plate, so there was a lot of background noise on the plate when absorbance was read.
  • This was especially seen in the blank control wells that should have had an absorbance reading of 0, but instead had some high readings of 0.3-0.5.
  • Most samples in plate 1 had high absorbance readings, while most samples in plate 2 had very low absorbance readings even though samples on both plates had the same native ferritin concentrations and the concentrations of peptide added were the same as well.
  • In addition, it was found that the constructed standard curve of absorbance vs concentration was not linear.

Future ELISA Experiments:

  • Use PBS Casein blocking buffer.
  • After each incubation, wash more thoroughly to prevent any background noise in absorbance readings.
  • Load ferritin at concentrations more similar to average human salivary ferritin levels.
  • Try different peptide incubation times to evaluate how quick peptide can bind to ferritin.
  • Perform ELISA using DNA aptamer with biotin conjugate.

DNA Trial 1 Results:

Results Viewed in Regular Lighting:

  • Image of the gel before viewed under UV light.
  • Bands are visible due to loading dye.
  • 2 bands are visible in the native ferritin + DNA samples.

DNA-trial-1-daylight-results

DNA Shift Assay Results Viewed in Regular Lighting

Results Viewed in UV Lighting:

  • Image of gel under UV light.
  • The results of this experiment might be successful, but additional runs need to be done to confirm.
  • Second band did not show under UV light for the ferritin/UV complexes → the second band is most likely DNA that did not bind to ferritin, and since there is less DNA that did not bind to ferritin it did not show up under the UV light.
  • The lane with ferritin loaded by itself (40ug) did not show up on the gel → this might be due to the ethidium bromide not effectively binding to ferritin which prevents UV detection of ferritin.
  • The lane with DNA by itself did show up on the gel and is represented by one band. Therefore, we know that the ethidium bromide did successfully bind to the DNA oligo which allowed for UV detection.
  • The samples of ferritin/DNA complexes produced 1 band in the gel. Since ferritin by itself was not able to be detected and DNA was able to be detected it is likely that for lanes 3 through 6 (samples that contained both ferritin and DNA), DNA was able to successfully bind to ferritin and that is why it showed up on the gel.
  • It is also possible the bands seen in lanes 3-6 are just DNA by itself and ferritin cannot be seen. However, lane 1 shows a pure DNA band that traveled further down the gel compared to the bands shown in lanes 3-6, so it is possible that these bands did not travel as far because DNA was bound to ferritin, and a ferritin DNA complex is bigger than DNA by itself.
  • Furthermore, the second band shown in lanes 3-6 in the image taken before the gel was viewed under UV light could show that there some DNA was bound to ferritin, and some did not bind (possibly due to an excess concentration of DNA added compared to ferritin).

DNA-trial-1-UV-results

DNA Shift Assay Results Viewed in UV Lighting

Future DNA Shift Assay Experiments:

  • Load various concentrations of the DNA aptamer on its own to understand the migration pattern of this oligo.
  • Load various concentrations of ferritin to understand migration pattern.
  • Run a gel with a dye that allows for clear visualization of both ferritin and DNA aptamer.
  • Run another gel with a dye known to bind to DNA only and not protein so that only ferritin with bound DNA can be visualized in the gel.
  • Load ferritin + DNA samples at different concentrations.
  • These future experiments will give us more data to confirm binding of the DNA aptamer to native ferritin.