Ponceau Stain Essay

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Figure 1 - Ponceau Stain blot
Key :
1) Protein Molecular Weight Marker
2) Recombinant pET41(+) EGFP plasmid (positive control)
3) Non-recombinant pet41(+) EGFP plasmid (negative control)
4) Green colored clone properly expresses EGFP (green)
5) White clone that does not express EGFP (red)
6) Unknown sample (blue)
7) Purified GST-EGFP control



A Ponceau stain can bind and identify all proteins. Lanes 2, 3, and 4 (our recombinant, nonrecombinant and green colony, respectively) have a slightly smeared pattern of multiple bands that goes from 245 kDa to 80 kDa. Lanes 2 and 4 have faint banding patterns that descend from 80 kDa downwards. Lane 3 ends a bit early, around the 135 kDa mark. Lanes 5-7 (our white colony, unknown colony and purified …show more content…

The Standards, their concentrations and their absorbance are listed in the table below the graph. The x-axis is the concentration of the Bovine Serum Albumin, while the y-axis is the absorbance reading that was received when the spectrometer was set to 595nm.

Discussion

The data confirms that EGFP is present, but in abnormally small and low amounts. The Ponceau Stain confirmed that there are proteins in each sample, and the Western Blot confirmed the presence of EGFP in the samples that should have EGFP and confirmed the absence of EGFP in the samples that should not have EGFP. The unknown sample was revealed to have EGFP, therefore confirming its identity as containing a recombinant plasmid. However, the UV illumination post-affinity chromatography and the low readings on the absorbance meter indicates that EGFP is just barely present. With EGFP composing of ~17% of the crude lysate, it is possible that proteins are in very small quantities and thus could not be expressed definitively. The source of this error was more likely in the beginning of the lab, but the error cannot be traced with certainty as the original gels for ligations, transformations and digests were penetrated and could not yield at a discernable …show more content…

The UV illumination after purification is faint and completely muted and the Bradford Assay absorbance readings were inconsistent in terms of pattern but still exceptionally small, which might contribute to the inability to get a precise reading. If the experiment was relaunched, samples with this small amount of EGFP protein should at first be checked without dilutions to see a rough estimation of how much raw protein is present. Had there’d been enough EGFP protein to create a successful result, elutions and washes 3 and 4 would glow considerably, along with the supernatant and flow through. All the elutions would also be brighter and more green. The absorbance readings and, as a result, the rest of the numbers, would also be higher and not fluctuate, especially as the frequencies were read for the last of the washes and the

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