Cyril Erica, Nicolette Nuñez 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

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INTRODUCTION

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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. 100µL of the BSA stock served as the base concentration. The following

procedure was done in order to prepare the different standard BSA concentrations: 20µL of diluent

water was added to 80µL of stock BSA to make the 800 BSA concentration, 40µL of water to 60µL

of stock BSA to prepare the next concentration, 80µL of water to 20µL of stock BSA for 200 BSA

concentration and lastly, 100µL of water and no stock BSA for the 0 concentration.

The protein extracted from the glass fish was also prepared in triplicates, with 100µL of the

sample per well. Next, 100µL 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

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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, 10µL 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.

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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.

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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.

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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 β-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.

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ENDNOTES

1. 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.

2. Ehrenberg, M. The Green Fluorescent Protein: Discovery, Expression and Development. The

Royal Swedish Academy of Sciences: September 30, 2008.

3. Gong, Z., Ju, B. and Wan. H. Green Fluorescent Protein (GFP) transgenic fiah and their

applications. Genetica 111: 213-225, 2001.

4. Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of

bacteriophage T4. Nature 227, 680-685, 1970.

5. 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: 201-207, 2002.

6. Tsien, R.Y. The Green Fluorescent Protein. Annual Reviews Biochemistry: 1998. 67: 509-44

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LIST OF FIGURES AND TABLES

Figure 1 The Glass Fish, Chanda ranga

Figure 2 The graph of the equations when the researchers plotted the absorbance

versus the Bradford Assay concentrations using the standard Bovine Serum

Albumin

Figure 3 The SDS-Page Result on the Agarose Gel

Figure 4 The Standard Curve used for the SDS-Page Analysis of GFP and BFP

Figure 5 The Western Blot Nitrocellulose Blot

Figure 6 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)

TRIALS

1 2 3

0 0.049 0.038 0.043

200 0.053 0.054 0.053

400 0.068 0.065 0.066

600 0.08 0.081 0.09

800 0.106 0.099 0.097

1000 0.114 0.116 0.099

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Table no. 2 – The SDS Page Rf Values

Table no. 3 – kDa Results of the Sample and Unknown

Y kDa

Unknown 4.85 71.54

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kDa log Mn Distance (mm) Rm (mm/mm2)

205 5.311754 23 0.378

116 5.064458 34 0.56

97 4.986772 38 0.62

84 4.924279 41 0.67

66 4.819544 45 0.74

55 4.740363 50 0.82

45 4.653213 55 0.90

36 4.556303 60 0.98

Unknown 44 0.72

GFP - Band 1 55 0.90

GFP – Band 2 59 0.97

BFP – Band 1 49 0.80

BFP – Band 2 56 0.92

GFP – Band 1 4.65 44.51

GFP – Band 2 4.57 37.45

BFP – Band 1 4.76 57.66

BFP – Band 2 4.63 42.63

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