Cyril Erica, Nicolette Nuez and Mariflor Rabe

Institute of Biology, College of Science University of the Philippines Diliman, Quezon City 1101 Philippines

Running Title: Green Fluorescent Protein on Glass Fish (Chanda ranga)

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS A. Protein Extraction and Quantitation using Bradford Protein Assay: To extract green fluorescent protein from the glassfish, Chanda ranga, 5 to 10mL of blood was collected from the green colored band on the sample fish obtained from the Bio 150 Laboratory Room. The collected blood was allowed to coagulate and was then spun down in a centrifuge for 5 minutes at 1000x g. After spinning, the supernatant was collected from the sample and was then stored. Bradford protein assay was used as a standard for the quantitation of the protein collected from the glassfish. Different concentrations of the assay were prepared in triplicates in a 96-well flat bottom microplate. 100L of the BSA stock served as the base concentration. The following procedure was done in order to prepare the different standard BSA concentrations: 20L of diluent water was added to 80L of stock BSA to make the 800 BSA concentration, 40L of water to 60L of stock BSA to prepare the next concentration, 80L of water to 20L of stock BSA for 200 BSA concentration and lastly, 100L of water and no stock BSA for the 0 concentration. The protein extracted from the glass fish was also prepared in triplicates, with 100L of the sample per well. Next, 100L of Bradford Reagent was added to each of the standard and protein sample wells. A StatFax 100 Microplate Reader was used to read the absorbance of the proteins in 630nm wavelength. The absorbance reading of each standard concentration and sample was then graphed versus the BSA concentration in order to measure the linearity. The slope and Y-intercept from the graph was recorded and used to compute for the sample protein concentration, with the help of the linear regression equation, y=mx+b, where m stands for the slope of the curve, y for the average absorbance of the sample and b for the y-intercept.

B. SDS-PAGE and Nitrocellulose Blotting

SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, was employed in order to separate the different protein samples according to their electrophoretic mobility. The steps employed in the preparation of the SDS-PAGE buffers were as described by Laemmli [1] wherein the following components were mixed in a container: 4.2 mL of the monomer, 3.125 mL of separating gel buffer which was 1.5M Tris-HCl, 5 mL of distilled water, 0.075 mL of 10% APS, 0.125 mL of 10% SDS and 0.025 mL TEMED. This was mixed gently and transferred into the gel cassette to about 2cm below the rim of the glass plate. The gel was overlayed with distilled water to allow the solution to polymerize. After 15 minutes, the water was then poured off and the resolving gel was then overlayed with a stacking gel which was prepared by mixing the following in a separate flask: 1.250 mL Monomer: stock acrylamide solution, 0.9375 mL of the stacking gel buffer: 1M Tris-HCl, 5.1375 mL distilled water, 0.075 mL 10% APS, 0.075 mL 10% SDS, and 0.025 mL TEMED. The well-forming comb was immediately placed after pouring the stacking gel over the resolving gel. Once the gel has polymerized, the comb was removed from the gel and the formed gel was installed to an electrophoresis apparatus. The electron chamber was then filled with the reservoir buffer solution. Before pouring in the protein samples to the wells, the samples were first diluted to a suitable protein concentration by adding an equal volume of double strength sample solubilization buffer solution. The samples were also heated in a boiling water bath for 5 minutes. Next, 10L of each of the samples were poured into the well. The protein sample included were the Bradford Assay which served as the standard, an unknown protein sample, blue fluorescent protein (BFP) and green fluorescent protein (GFP). The gels were then ran under 150 V in the electrophoresis apparatus in order to separate the bands of the sample proteins. After electrophoresis, the proteins were visualized by Coomassie blue staining. The gel bands were then excised and destained. Identification of the unknown protein sample was carried out by measuring its molecular weight from the bands. The molecular weight of the BSA standards, the distance of the bands from

the stacking gel and their correspong Rm were used in order to come up with equation of the line which was afterwards used to compute for the weight of the protein. Determination of the molecular weight of the unknown was determined by substituting X to the equation y = mx+b. After electrophoresis, the proteins were electroblotted to a Triton-free nitrocellulose membrane in a transfer buffer. Afterwards, staining and destaining were also carried out.

RESULTS

The researches obtained different absorbances at different concentrations of the standards in Bradford Assay. [2] Figure 2 shows the graph of the equations when we plotted the absorbance versus the Bradford Assay concentrations. The obtained equation of the line, y = 8E-05x+0.036, with the regression coefficient closest to 1 was used in order to compute for the green fluorescent protein (GFP) and blue fluorescent protein (BFP) concentrations. The researchers obtained a concentration of ______ and _______, respectively. When the GFP that was extracted from the glassfish was made to run in an SDS-PAGE, along with BFP, an unknown protein and the BSA standards, the researchers obtained these bands. [Figure 3] The molecular weight of the unknown protein sample was measured for its identification. This was done with the use of the distances of the bands from the stacking gel layer. Determination of the molecular weight of the unknown was determined by substituting X from the equation of the line, y = -1.143(x) + 5.679. Researchers found out that the molecular weight of the proteins were as follows: unknown protein: 71.54kDa, ______: 44.5 kDa, ______: 37.45 kDa, ________: 57.66 kDa, and _____42.63: kDa. Blotting the gel in a nitrocellulose membrane, meanwhile, gave the researchers the following bands. [5] Protein molecular weight determination was not employed in this step.

DISCUSSION

The researches learned that this has been the first time that Green Fluorescent Protein has been studied on Chandra ranga, also known as Glass Fish. Bradford Assay is to determine the Molecular Weight of the GFP based on the knowledge that the structure is of -barrel [2] with 238 amino acids [1]. Blue Fluorescent Protein/Cyan Fluorescent Protein has been determined the same time as the GFP. It has been determined that GFP present in the glass fish half the amount compared to the BFP present in the Glass Fish. GFP and BFP express two distinct bands on the agarose gel during electrophoresis. The two fractions of both proteins are near each other which were conclusive that the two proteins differ only on some amino acid chains. The researchers hypothesized that the fluorescing protein and the color giving protein is subdivided on the two distinct fractions.

ENDNOTES 1. 2. 3. 4. 5. Alberts, B., Johnson, A., Lewis, L., et. al. Chapter 8: Manipulating Protiens, DNA and RNA. The Molecular Biology of the Cell, 5th Edition. Garland Science: 2008. Ehrenberg, M. The Green Fluorescent Protein: Discovery, Expression and Development. The Royal Swedish Academy of Sciences: September 30, 2008. Gong, Z., Ju, B. and Wan. H. Green Fluorescent Protein (GFP) transgenic fiah and their applications. Genetica 111: 213-225, 2001. Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685, 1970. Molina, A. Carpeaux, R. Martial, J.A., Muller, M. A Transformed fish cell line expressing a green fluorescent protein-luciferase fusion gene responding to cellular stress. Toxicology In Vitro 16: 201207, 2002. 6. Tsien, R.Y. The Green Fluorescent Protein. Annual Reviews Biochemistry: 1998. 67: 509-44

LIST OF FIGURES AND TABLES Figure 1 Figure 2 The Glass Fish, Chanda ranga The graph of the equations when the researchers plotted the absorbance versus the Bradford Assay concentrations using the standard Bovine Serum Albumin Figure 3 Figure 4 Figure 5 Figure 6 The SDS-Page Result on the Agarose Gel The Standard Curve used for the SDS-Page Analysis of GFP and BFP The Western Blot Nitrocellulose Blot The Agarose Gel used for Western Blot analysis

Table 1 Results of the Bradford Assay Analysis of Proteins Table 2 Results of the Absorbance and computed Molecular Weight of Proteins Table 3 The Molecular Weights of the unknown and the Analytes GFP and BFP

Figure no. 1: The Glass Fish, Chanda ranga

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Figure no. 2 - Bradford Assay concentration vs Absorbance Graph of Bovine Serum Albumin

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Figure no. 3 SDS-Page Agarose Gel with Bands

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Figure no. 4 SDS-Page Standard Curve

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Figure no. 5 Nitrocellulose Membrane Blot

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Figure no. 6 The Agarose Gel used for Western Blot

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Table no.1 The Bradford Assay Absorbance Readings

BSA Concentration (in ug/mL) 0 200 400 600 800 1000 1 0.049 0.053 0.068 0.08 0.106 0.114

TRIALS 2 0.038 0.054 0.065 0.081 0.099 0.116 3 0.043 0.053 0.066 0.09 0.097 0.099

Table no. 2 The SDS Page Rf Values

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kDa 205 116 97 84 66 55 45 36

log Mn 5.311754 5.064458 4.986772 4.924279 4.819544 4.740363 4.653213 4.556303 Unknown GFP - Band 1 GFP Band 2 BFP Band 1 BFP Band 2

Distance (mm) 23 34 38 41 45 50 55 60 44 55 59 49 56

Rm (mm/mm2) 0.378 0.56 0.62 0.67 0.74 0.82 0.90 0.98 0.72 0.90 0.97 0.80 0.92

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Table no. 3 kDa Results of the Sample and Unknown Y 4.85 4.65 4.57 4.76 4.63 kDa 71.54 44.51 37.45 57.66 42.63

Unknown GFP Band 1 GFP Band 2 BFP Band 1 BFP Band 2

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