Discussion
Introduction Materials & Methods Results Literature Cited
The results obtained in this experiment following Western Blot Analysis and SDS PAGE were inconclusive. There was no data confirming the presence of the Rubisco Large Subunit protein in any of the designated samples.
The protein bands visualized in the SDS PAGE gel were not positive for the 55 kDa Rubisco Large Subunit. Due to the blurry nature of the protein bands, we were not able to confirm the presence of Rusbisco for any sample. This may be attributed to the partial degradation of protein samples. In order to obtain significant results, another SDS gel should be run containing smaller amounts of purified protein; a smaller concentration may enable more definitive bands to be visualized. Also, a different stacking gel may also be utilized to create greater compression of protein prior to entering the separating gel thus reducing the occurrence of blurred results. Finally, running the gel for a shorter time period may increase protein visualization; running our gel for too long may have caused the protein to run off of the gel. (Figure 2A)
The Western Blot gel resulted in no visualization in any protein. Due to lack of specific binding there was no way to confirm the presence of the Rubisco protein in any sample. These results may have been caused by improper protein transfer from previously visualized proteins from the SDS PAGE gel to the nylon membrane. The lack of protein detection may potentially be attributed to the inability of the primary or secondary antibodies to bind the Rubisco protein (assuming successful protein transfer and blocking). However, another group obtained visible bands on their portion of the western blot membrane. This fact decrease the likelihood that the antibodies were of low affinity. In order to increase the binding of primary antibody to the Rubisco protein, it may be necessary to electroblot for a longer period of time, as larger proteins transfer to membranes more slowly than smaller proteins.
In order to support our hypothesis that inorganic plants contained higher levels of the Rubisco Large Subunit Protein, due to increased nutritional supplementation via fertilizers, conclusive data would be necessary. Significant data on both SDS PAGE and Western Blot analysis would need to illustrate the presence of Rubisco at 55kDa and specific antibody binding respectively at increased levels in inorganic plants in comparison with their organic counterparts. Inconclusive data was obtained from the performed experiment resulting in the absence of support for or against the initial hypothesis.
The Real-time DNA data was also inconclusive. Only the inorganic onion DNA samples were successfully amplified (Figure 3A). Confirmation of inorganic onion DNA amplification can be observed in the agarose gel (Figure 4). Even though the inorganic onion DNA was successfully amplified, the temperature of melting per sample was variable leading us to believe that the quality of DNA was of low purity. Impure DNA may have resulted from contamination of samples. Unsuccessful amplification of sample DNAs were most likely due to grossly inadequate amounts of DNA in initial PCR reaction tubes. These discrepancies may be attributed to inaccurate concentration readings provided by the nanodrop machine. To remedy this error, spectroscopy should be utilized to obtain more accurate DNA concentration values. Repetition of Real-time PCR with DNA samples of accurate concentrations and of higher purity may provide improved data. Based on current results no conclusions for the acceptance or negation of the initial hypothesis may be made.
In the future, it will be helpful to apply the above suggestions to obtain significant results or at the very least to diagnose previous error. In doing so, a greater understanding of Rubisco content may be achieved for desired samples. Optimistically, this experiment has contributed to the potential understanding of environmental influences on Rubisco expression. More insight may be provided upon further evaluation of Rubisco in organic and inorganic plants based upon fertilization techniques.