Separating plant pigments in Papaver somniferum using Chromatography and SDS PAGE Chelsea Parrocha, Polaris K-12 BioPREP

This projectʼs focus and was to find the ratioʼs of different proteins which contributed to a flowerʼs color by the means of chromatography—and assuming that flower pigment is a protein. The flower used for this experiment was Poppies due to the wide variety of colors they can express. Once samples were extracted from the chromatography, each sample would run through an SDS PAGE and from there create a standard ladder for flower colors based on the proteinʼs size. However, none of the methods of separating proteins did not produce successful results. Through this project, it was proven that flower pigments were not proteins. After some research, it was discovered that flower pigments are essentially anthocyanins—plant secondary metabolites. An additional test was performed on poppy flower pigment to test if there were any traces of protein. Under a Bradford Assay, it was proven that there were minuscule traces of proteins in flower pigment. In conclusion, flower pigments are not proteins and cannot be separated by methods used for separating proteins.

Introduction:

Methods:

Results: • The tests that were performed in this project were Size Exclusion Chromatography, HIC Chromatography and SDS PAGE • Bradford Assay was also used as an additional test • SDS PAGE did not work at all—there was nothing in the wells that were loaded with the six different poppy samples.

Discussion:

Graphic Graphic

• SDS PAGE and Size Exclusion Chromatography did not provide successful results •  HIC worked to some extent, but in terms of separating the proteins to find a blending ratio, it was not successful as well. • The hypothesis that flower pigments are proteins—and therefore can be sorted out with methods used to separate proteins, turned out to be untrue • Proteins are actually Anthocyanins (according to James Shield). • Anthocyanins—a type of flavonoid • Positive and negative charges in the Aryl-chroman ring system is what gives color to plant pigments • It is possibly this reason why HIC worked as it did • High-pressure Liqud Chromatography (HPLC) was used on anthocyanins and its ph was measured mircospectrophotometrically

Laemmli, Ulrich K. "The Cleavage of Structural Proteins During the Assembly of the Head of the Bacteriophage T-4." Nature 227 (1970): 680 - 685. Web. 8 May 2012. <http://bioinfcpcri.org/misc/nature.pdf>. Bradford, Marion M. "A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding."Analytical Biochemistry 72 (1976): 248-254. Web. 8 May 2012. <http://hoffman.cm.utexas.edu/courses/bradford_assay.pdf>. Griesbach, R. J. "The Inheritance of Flower Color in Petunia hybrida Vilm." The Journal of Heredity (1996): 241-245. Web. 8 May 2012. <http://jhered.oxfordjournals.org/content/87/3/241.short>. Tennikova, Tatiana B., and Svec Frantisek. "High-performance membrane chromatography: Highly efficient separation method for proteins in ion-exchange, hydrophobicinteraction and reversed-phase modes." ScienceDirect.com (2001): Web. 8 May 2012. <http://http://www.sciencedirect.com/science/article/pii/002196739383340X/doi/abs/10.1021/ac00242a025>. Griesbachv, Gary L. "Size Exclusion Chromatography." Analytical Chemistry(1982): 265R–276R. Web. 8 May 2012.<http://pubs.acs.org/doi/abs/10.1021/ac00242a025>.

Bibliography:

Collect Flowers • Poppies were the perfect sample to use due to the wide variety of colors they can express •  6 poppies collected on the same day and were stored in the fridge until further use

Grind to make slurry • Took petals of poppies with and grinded it with a pestle and mortar

Separate Proteins • Both chromatography’s had five fractions per test tube

HIC • Separated based off of protein's Hydrophobocity

Size Exclusion • Separated based on protein size

SDS PAGE • Separate proteins by size latter and compare with a standard

Collect fractions and compare which chromatography works better. Whichever method works better, the sample from that method will be used in SDS PAGE and further analysis.

Qualitative Analysis # of colors per flower Color (visually)

Figure 1

Sample Observation

Red For some reason, the colors from the collection turned out to be a Dimetapp cough syrup purple.

Highlighter Red

This sample was done twice. Times, the samples turned out to be a light champagne color.

Fuchsia The sample looked like it would work, but the collection was just a dilute pink.

Samples Observation

Red The same Dimetapp purple occurred with HIC. At the end, there was a blue ring that stuck at the top of the column (Figure 9.) Once the sample was extracted, it was a dark purple (Figure 10)

Highlighter Red

HIC did not work as well with this sample. The collections were very dilute.

Fuchsia Just like the Red Sample, a darker color ring occurred at the top of the column. However, the collection were just dilutions.

Size Exclusion Chromatography HIC Chromatography

Figure 2

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Figure 9

Figure 4

Abstract

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Figure 10