FMS 1 EXPERIMENT REPORTPROTEOMIC SESSION

Name: NAINA KARAMINA SAKINANIM : 07120100102Group: B2-3

FACULTY OF MEDICINEUNIVERSITAS PELITA HARAPAN2010ABSTRACT

Protein is a crucial and essential component of all body tissues. Protein is crucial and essential for body because it contains the essential amino acids that was needed by bodies for their metabolic process. Protein also can help bodies to generate such big number of new cells with their useful components, so no wonder if the protein is often said as a crucial and essential component for the body because every cell in the body is continuously being recycled or being renewed in order to reach the homeostasis in the body. Because of the protein is very important for the body, it also would be important to study about this amino acids. We have to study about protein because we have to know what the protein really is, what the source of protein, what are the components that exist in the protein, and many more. One of the benefit of studying the protein is we can know the advantages and the disadvantages of the protein. Because of that, several experiments were done in this proteomic session in order to know more about the proteins in the human body. Those experiments were used the blood as their sample and they have several purposes, as to determine the concentration of the protein in the sample, to determine the appropriate dilution factor of the sample, to determine the molecular mass of certain proteins in total serum protein, to determine the distribution of proteins among fractions, to get the quantitative determination of the antigen AFP concentration in human serum, and also to measure blood glucose level from serum sample. Those experiments were essential because by doing those experiments we can know the amount of the protein that exist in our body, so it can prevents from several disease that can happen as a result of the deficiency or the excessive amount of the protein in the body.

List of Figures

1. Figure 1. Primary Protein Structure71. Figure 2. Secondary Protein Structure71. Figure 3. Hydrogen Bond in Tertiary Protein Structure81. Figure 4. Tertiary Protein Structure81. Figure 5. Quaternary Protein Structure9

ivContents

ABSTRACTiiList of FiguresiiiList of TablesivContentsvChapter 1 INTRODUCTION1Chapter 2 MATERIALS AND METHODS32.1. Materials32.1.1. Bradford Test32.1.2. Protein Separation Through SDS-PAGE32.1.3. Enzyme-Linked Immunosorbent Assay32.1.4. Colorimetric Determination of Blood Sugar Level42.2. Methods42.2.1. Bradford Test42.2.2. Protein Separation Through SDS-PAGE52.2.3. Enzyme-Linked Immunosorbent Assay62.2.4. Colorimetric Determination of Blood Sugar Level6Chapter 3 RESULTS83.1. Bradford Test83.2. Protein Separation Through SDS-PAGE83.3. Enzyme-Linked Immunosorbent Assay93.4. Colorimetric Determination of Blood Sugar Level10Chapter 4 DISCUSSION12REFERENCES15

Chapter 1INTRODUCTION

In the experiments in this proteomic session, the main substance that was used is protein. Protein is a large molecule composed of one or more chains of amino acids in a specific order determined by the base sequence of nucleotides in the DNA coding for the protein. Protein is something the body both is and cannot remain without. Proteins are required for the structure, function, and regulation of the body's cells, tissues, and organs. Each protein has unique functions. Proteins are essential components of muscles, skin, bones and the body as a whole. Examples of proteins include whole classes of important molecules, among them enzymes , hormones, and antibodies. Protein is one of the three types of nutrients used as energy sources by the body, the other two being carbohydrate and fat. Proteins and carbohydrates each provide 4 calories of energy per gram, while fats produce 9 calories per gram. The word "protein" was introduced into science by the great Swedish physician and chemist Jns Jacob Berzelius (1779-1848) who also determined the atomic and molecular weights of thousands of substances, discovered several elements including selenium, first isolated silicon and titanium, and created the present system of writing chemical symbols and reactions. Protein is the main component of muscles, organs, and glands. Every living cell and all body fluids, except bile and urine, contain protein. The cells of muscles, tendons, and ligaments are maintained with protein. Children and adolescents require protein for growth and development.[footnoteRef:-1] [-1: http://www.medterms.com/script/main/art.asp?articlekey=6554]

There are three different structures of protein which is the primary structure, the secondary structure, the tertiary structure, and the quaternary structure. The primary structure of peptides and proteins refers to the linear number and order of the amino acids present. The convention for the designation of the order of amino acids is that the N-terminal end (i.e. the end bearing the residue with the free -amino group) is to the left (and the number 1 amino acid) and the C-terminal end (i.e. the end with the residue containing a free -carboxyl group) is to the right.

Figure 1in the secondary structure, there are 2 typical shapes that have been develop which is coils (an alpha helix) or folds (beta pleated sheets)

Figure 2

The -HelixThe -helix is a common secondary structure encountered in proteins of the globular class. The formation of the -helix is spontaneous and is stabilized by H-bonding between amide nitrogens and carbonyl carbons of peptide bonds spaced four residues apart. This orientation of H-bonding produces a helical coiling of the peptide backbone such that the R-groups lie on the exterior of the helix and perpendicular to its axis.-Sheets-sheets are composed of 2 or more different regions of stretches of at least 5-10 amino acids. The folding and alignment of stretches of the polypeptide backbone aside one another to form -sheets is stabilized by H-bonding between amide nitrogens and carbonyl carbons. -sheets can be depicted in ball and stick format or as ribbons in certain protein formats.Some proteins contain an ordered organization of secondary structures that form distinct functional domains or structural motifs. Examples include the helix-turn-helix domain of bacterial proteins that regulate transcription and the leucine zipper, helix-loop-helix and zinc finger domains of eukaryotic transcriptional regulators. These domains are termed super-secondary structures.Tertiary structure refers to the complete three-dimensional structure of the polypeptide units of a given protein. Included in this description is the spatial relationship of different secondary structures to one another within a polypeptide chain and how these secondary structures themselves fold into the three-dimensional form of the protein. Secondary structures of proteins often constitute distinct domains. Therefore, tertiary structure also describes the relationship of different domains to one another within a protein. The interactions of different domains is governed by several forces: These include hydrogen bonding, hydrophobic interactions, electrostatic interactions and van der Waals forces.

Figure 3 Figure 4

The quaternary structure is the structure formed by monomer-monomer interaction in an oligomeric protein. Oligomeric proteins are proteins with multiple polypetide chains that are held in association by the same non-covalent forces that stabilize the tertiary structures of proteins. Oligomeric proteins can be composed of multiple identical polypeptide chains or multiple distinct polypeptide chains. Proteins with identical subunits are termed homo-oligomers. Proteins containing several distinct polypeptide chains are termed hetero-oligomers.Hemoglobin, the oxygen carrying protein of the blood, have more than one string of amino acids which are then hooked together in this structure. It contains two and two subunits arranged with a quaternary structure in the form, 22. Hemoglobin is, therefore, a hetero-oligomeric protein.[footnoteRef:0] [0: http://themedicalbiochemistrypage.org/protein-structure.html]

Figure 5

The first experiment in this proteomic session is the Bradford test. The Bradford protein assay or Bradford test is one of several simple methods commonly used to determine the total protein concentration of a sample. The method is based on the proportional binding of the dye Coomassie to proteins. Within the linear range of the assay (~5-25 mcg/mL), the more protein present, the more Coomassie binds. Furthermore, the assay is colorimetric; as the protein concentration increases, the color of the test sample becomes darker. Coomassie absorbs at 595 nm. The protein concentration of a test sample is determined by comparison to that of a series of protein standards known to reproducibly exhibit a linear absorbance profile in this assay.[footnoteRef:1] [1: http://ww2.chemistry.gatech.edu]

Although different protein standards can be used, the most widely protein which is Bovine Serum Albumin (BSA) was used in this experiment.The purpose of this experiment are to determine the concentration of the protein and the appropriate dilution factor of the solution. The solution that was used in this experiment is the serum from the blood sample. Serum is the component of the blood that have similarity in composition with plasma, but lacks the coagulation factors. Serum and plasma can be gotten from the centrifugation process of the blood sample that is not been added with the anticoagulant. If this sample being mixed, only serum separates with other components (plasma already mix with other components). Blood plasma is the yellowish liquid component of blood in which blood cells are normally suspended and various substances like food, waste products, hormones and gases remain in solution. It transports everything except fat droplets and, in vertebrates, oxygen.It makes up about 55 percent of the blood by volume and is composed primarily of water and proteins.It composed about 92 percent water with 6.5 percent proteins. The remaining components are 0.8 percent salts, 0.6 percent lipids and 0.1 percent glucose. Blood plasma contains salts, hormones, and proteins. There are two major groups of protein in blood plasma, which is albumin and immunoglobulins. Albumin has the purpose to keep blood from leaking out of blood vessels and to bind certain hormones, while immunoglobins involved in actively defending the body against invaders such as bacteri and fungi. It also help clot the blood. The primary function of blood plasma is to serve as a reservoir for water. It supplies water to cells that are dehydrated and also absorbs excess water from cells.[footnoteRef:2] [2: http://www.ehow.com/about_5502288_blood-plasma-regulations.html]

The second experiment that was done is Protein Separation Through SDS-PAGE. This experiment has the purpose to determine the molecular mass of certain proteins in total serum protein and the distribution of proteins among fractions. SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) is a very common method for separating protein according to their electrophoretic mobility by electrophoresis (migration of charged molecules in solution in response to an electric field) uses a discontinuous polyacrylamide gel as a support medium and Sodium Dodecyl Sulfate (SDS) to denature the protein. SDS is an anionic detergent which denatures proteins by "wrapping around" the polypeptide backbone and SDS binds to proteins fairly specifically in a mass ratio of 1.4:1. In so doing, SDS confers a negative charge to the polypeptide in proportion to its length, example: the denatured polypeptides become "rods" of negative charge cloud with equal charge or charge densities per unit length. It is usually necessary to reduce disulphide bridges in proteins before they adopt the random-coil configuration necessary for separation by size. In separating and stacking gel, we use TEMED (tetramethylethylenediamine) which its function as a catalyst in the presence of free radical initiatiors to accelerate the copolymerization of acrylamide and diacetone acrylamide into PAD. In denaturing SDS-PAGE separations therefore, migration is determined not by intrinsic electrical charge of the polypeptide, but by molecular weight. Determination of Molecular Weight is done by SDS-PAGE of proteins of known molecular weight along with the protein or nucleic acid to be characterised. A linear relationship exists between the logarithm of the molecular weight of an SDS-denatured polypeptide, or native nucleic acid, and its Rf. The Rf is calculated as the ratio of the distance migrated by the molecule to that migrated by a marker dye-front. A simple way of determining relative molecular weight by electrophoresis (Mr) is to plot a standard curve of distance migrated vs. log10MW for known samples, and read off the logMr of the sample after measuring distance migrated on the same gel.[footnoteRef:3] [3: http://www.mcb.uct.ac.za/sdspage.htm]

in this second experiment, there are six steps that required in order to reach the goal. The steps are preparation of gels, casting of separating gel, casting of stacking gel, fractionation of serum protein by ethanol precipitation, preparation of samples for SDS-PAGE, and staining and destaining the gel. In the fourth step, the purpose of the step is to fractionate protein by precipitation. Precipitation is widely used in downstream processing of biological products, such as proteins. This unit operation serves to concentrate and fractionate the target product from various contaminants. For example, in the biotechnology industry protein precipitation is used to eliminate contaminants commonly contained in blood. Academic research on protein precipitation explores new protein precipitation methods. The underlying mechanism of precipitation is to alter the solvation potential of the solvent and thus lower the solubility of the solute by addition of a reagent.[footnoteRef:4] [4: http://en.wikipedia.org/wiki/Protein_precipitation]

Precipitation can be done by using several solvents. One of the solvent that can be used in the precipitation process is the organic solvents. In the second experiment we used the ethanol which is the organic solvent for the precipitation process.Addition of miscible solvents such as ethanol or methanol to a solution may cause proteins in the solution to precipitate. The solvation layer around the protein will decrease as the organic solvent progressively displaces water from the protein surface and binds it in hydration layers around the organic solvent molecules. With smaller hydration layers, the proteins can aggregate by attractive electrostatic and dipole forces. Important parameters to consider are temperature, which should be less than 0 C to avoid denaturation, pH and protein concentration in solution. Miscible organic solvents decrease the dielectric constant of water, which in effect allows two proteins to come close together. At the isoelectric point the relationship between the dielectric constant and protein solubility is given by:

S0 is an extrapolated value of S, e is the dielectric constant of the mixture and k is a constant that relates to the dielectric constant of water. The Cohn process for plasma protein fractionation relies on solvent precipitation with ethanol to isolate individual plasma proteins.a clinical application for the use of methanol as a protein precipitating agent is in the estimation of bilirubin.[footnoteRef:5] [5: http://en.wikipedia.org/wiki/Protein_precipitation]

The third experiment that was done is the enzyme-linked immunosorbent assay which has the purpose to get the quantitative determination of the antigen AFP concentration in human serum.The last experiment in this proteomic session is the colorimetric determination of blood sugar level. This experiment was done in order to measure the blood glucose level from serum sample. Blood glucose, is the bodys fuel that feeds the brain, nervous system, and tissues. A healthy body makes glucose not only from ingested carbohydrates, but also from proteins and fats, and would not be able to function without it. Maintaining a balanced blood glucose level is essential to a bodys everyday performance. Glucose is absorbed directly into the bloodstream from the intestine and results in a rapid increase in the blood glucose level. The pancreas releases insulin, a natural hormone, to prevent blood glucose levels from excessively elevating, and aids in the moving of glucose into the cells. Glucose is then carried to each cell, providing them with the energy needed to carry out its specific function. Healthy blood glucose levels are considered to be in the 70-120 range. One high or low reading does not always indicate a problem, but the glucose level should be monitored for 10-14 days. There are several different tests that can be administered to determine whether an individual has a problem maintaining a normal glucose level such as: a fasting blood sugar test, an oral glucose test, or a random blood sugar test.[footnoteRef:6] In this experiment, the blood glucose assay that was done is the assay by using the colorimeter or spectrophotometer. A spectrophotometer or colorimeter makes use of the transmission of light through a solution to determine the concentration of a solute within the solution. A spectrophtometer differs from a colorimeter in the manner in which light is separated into its component wavelengths. A spectrophotometer uses a prism to separate light and a colorimeter uses filters. Both are based on a simple design of passing light of a known wavelength through a sample and measuring the amount of light energy that is transmitted. This is accomplished by placing a photocell on the other side of the sample. All molecules absorb radiant energy at one wavelength of another. Those that absorb energy from within the visible spectrum are known as pigments. Proteins and nucleic acids absorb light in the ultraviolet range. The following figure demonstrates the radiant energy spectrum with an indication of molecules which absorb in various regions of that spectrum. [6: http://www.wisegeek.com/what-is-blood-sugar.htm]

The design of the single beam spectrophotometer involves a light source, a prism, a sample holder and a photocell. Connected to each are the appropriate electrical or mechanical systems to control the illuminating intensity, the wavelength, and for conversion of energy received at the photocell into a voltage fluctuation. The voltage fluctuation is then displayed on a meter scale, is displayed digitally, or is recorded via connection to a computer for later investigation.Spectrophotometers are useful because of the relation of intensity of color in a sample and its relation to the amount of solute within the sample. For example, if you use a solution of red food coloring in water, and measure the amount of blue light absorbed when it passes through the solution, a measureable voltage fluctuation can be induced in a photocell on the opposite side. If now the solution of red dye is diluted in half by the addition of water, the color will be approximately 1/2 as intense and the voltage generated on the photocell will be approximately half as great. Thus, there is a relationship between the voltage and the amount of dye in the sample.Given the geometry of a spectrophotometer, what is actually measured at the photocell is the amount of light energy which arrives at the cell. The voltage meter is reading the amount of light transmitted to the photocell. Light transmission is not a linear function, but is rather an exponential function. That is why the solution was approximately half as intense when viewed in its diluted form.We can however monitor the transmission level and convert it to a percentage of the amount transmitted when no dye is present. Thus, if 1/2 the light is transmitted, we can say that the solution has a 50% Transmittance. Note that it is always relative to a solution containing no dye. Transmittance is the relative percent of light passed through the sample. What makes all of this easy to use, however, is the conversion of that information from a percent transmittance to an inverse log function known as the Absorbance (or Optical Density).[footnoteRef:7] [7: http://homepages.gac.edu/~cellab/appds/appd-g.html]

Chapter 2MATERIALS AND METHODS

2.1. Materials2.1.1. Bradford Test1. Bovine Serum Albumin Standard Set (0.125, 0.25, 0.5, 0.75, 1, 1.5, 2 mg/ml)2. 1X Dye Reagent Vol. 1 ml3. Cuvettes2.1.2. Protein Separation Through SDS-PAGE1. Laemmli sample buffer2. Coomassie blue staining solution50% Methanol0.05% (v/v) Coomassie brilliant blue R-25010% (v/v) Acetic Acid40% H2O 3. TEMED2.1.3. Enzyme-Linked Immunosorbent Assay1. Rabbit anti-human AFP coated microtitter plate with 96 wells2. Zero buffer (13 ml)3. Reference standard set contain 0, 5, 20, 50, 150, and 300 mg/ml AFP, lyophilized4. 18 ml enzyme conjugate reagent5. 11 ml TMB reagent6. 11 ml 1N HCl stop solution7. Distilled water8. Microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-2 OD or greater at 450 nm wavelength.2.1.4. Colorimetric Determination Of Blood Sugar Level1. Otoluidine reagent2. Standard glucose solution 200mg/dL3. Distilled water4. Centrifuge5. Spectophotometer6. Cuvette7. Reaction tube8. Aluminium foil9. Water bath2.2. Methods2.2.1. Bradford Test20 l of each standard and unknown sample was pipetted into microcentrifuge tubes and each tube was added by 1 ml of 1X Dye reagent. Blank sample should be made using distilled water and dye reagent. The tubes were inverted in order to mix the solution. After that, the tubes were being incubated at room temperature for at least 5 minutes, it shouldnt be incubated longer than 1 hour at room temperature. Then, the spectrophotometer was set to 595 nm. The blank sample was used to zero the instrument and the absorbance of the standards and unknown samples were measured by using the spectrophotometer. 2.2.2. Protein Separation Through SDS-PAGE2.2.2.1. Preparation of gelsa 12% separating gel was prepared by adding 1.28 ml of Distilled water, 1.6 ml of 30% Acrylamide, 1.04 ml of 1.5 M Tris-HCL Buffer (pH 8.8), and 40 l of 10% SDS to a 50 ml beaker. For the stacking gel, a stacking gel was prepared by adding 0.7 ml of Distilled water, 0.165 ml of 30% Acrylamide, 0.125 ml of 0.5 M Tris-HCL Buffer (pH 6.8), and 10 l of 10% SDS to a 50 ml beaker.2.2.2.2. Casting of separating gelThe gel casting mould was assembled as instructed by the demonstrator. 80 l of 10% APS solution and 8 l of TEMED was added and swirled very gently for about 5 seconds. About 3.6 ml of separating gel solution was added carefully to avoid any spillages by using 1 ml automatic pipette. After that, the separating gel solution was overlayed immediately with distilled water. Then, the separating gel solution was left to polymerize for about 30 minutes and after it has been polymerized, the distilled water was poured out.2.2.2.3. Casting of stacking gel 20 l of 10% APS solution and 2 l of TEMED was added and swirled very gently for about 5 seconds. About 1 ml of stacking gel was also added on top of polymerized separating gel by using 1 ml automatic pipette and any bubbles were tried to be avoided. After that, the comb was placed between the glass plates with one end higher than the other and it was pressed down carefully so that the teeth are level about 5 mm from the top of the separating gel. If necessary, more stacking gel might be added to ensure that the sides of the well are complete. Then, the separating gel was left to polymerize for about 20-30 minutes, the comb was removed carefully and the wells were being washed out for at least 3 times with distilled water.2.2.2.4. Fractionation of serum protein by ethanol precipitation125 l of ice-cold ethanol and 250 l of serum were added to the 1.5 ml microcentrifuge tube and the tube was vortex and stored in ice bucket. After it was stored in ice bucket for 5 minutes, it was centrifuged for 3 minutes at 7000 rpm and 4C. After that, the supernatant was decanted carefully to another microcentrifuge tube, it was labeled Es and stored in the ice bucket. A towel paper was twisted and inserted into the microcentrifuge to draw off the last of the supernatant from above the precipitated proteins by capillary action. Then, 1 ml of chilled 50% ethanol was added to the precipitate to washed out the last traces of supernatant. The samples were resuspended by repeated aspiration with 1000 l automatic pipette to break up the pellet (this step should tried to be done quickly so that the preparation didnt heat up with the warmth of our fingers). 200 l of the samples was taken from the previous step and added to a new tube. Then, the tube was centrifuged again for 3 minutes at 7000 rpm and 4C, the wash supernatant was decanted into the plastic waste cup and the last drop was removed with a twisted towel paper. The Ep fraction was the precipitated one. In the end, the samples were stored in ice bucket.2.2.2.5. Preparation of samples for SDS-PAGE10 l of Es was taken out to another microcentrifuge tube and 45 l of sample buffer was added to it. After that, 90 l of sample buffer was added to the Ep fraction then it was vortex to mix the precipitate thoroughly with the sample buffer. The 1000 l automatic pipette could be use to break up the pellet if there was trouble resuspending the pellet. Then, Es and Ep tubes were put into a rack in the boiling water and they were boiled for 5 minutes (only the tips of the tubes need to be in contact with the boiling water). Next, the microcentrifuge tubes were removed from the boiling water bath and the exteriors were dried with a towel paper. The tubes then were centrifuged at room temperature for 3 minutes to pellet any insoluble materials that may have precipitated during boiling. After the centrifugation process was done, the samples were ready to be loaded into the appropriate wells of the gel. About 5-12 l of the samples was loaded to the gel. The precipitates shouldnt been stirred up before loading.2.2.2.6. Staining and destaining the gelThe gel was removed and stained with coomassie blue in wobble table for 1 hour. After that, the gel was destained with destain solution in wobble table for 1 hour and the destaining solution was changed with fresh one every 1 hour until background is clear. Then, the gel was wrapped in a piece of cellophane plastic.2.2.3. Enzyme-Linked Immunosorbent AssayThe desired number of coated wells in the holder was secured. 20 l of standard, specimens, and controls were dispensed into appropriate wells. After that, 100 l of zero buffers was also dispensed into each well and it was mixed thoroughly for 30 seconds (it was very important to have complete mixing in this setup). Then, it was incubated at room temperature (18-25C) for 30 minutes. After the incubation process was done, the incubation mixture was removed by flicking plate content into a waste container. Then, the microtiter wells were rinsed and flicked 5 times with distilled water. The wells were sharply struck onto absorbent paper or paper towel to remove all residual water droplets. 150 l of Enzyme Conjugate Reagent was dispensed into each well and gently mixed for 5 seconds. Next, it was incubated at room temperature for 30 minutes. After the incubation process was done, the incubation mixture was removed by flicking plate contents into a waste container. Then, the microtiter wells were rinsed and flicked 5 times with distilled water and they were sharply struck onto absorbent paper to remove residual water droplets. 100 l of TMB Reagent was dispensed into each well and gently mixed for 5 seconds. It was incubated at room temperature for 20 minutes. After the incubation process was done, 100 l of Stop Solution was added to each well to stop the reaction. Then, gently mixed for 30 seconds (make sure that all the blue color changes to yellow color completely). The optical density was read at 450 nm with a microtiter reader within 15 minutes.2.2.4. Colorimetric Determination Of Blood Sugar

2.2.4.1. Performing standar curve standard glucose solutions

The glucose standars 500 mg/dl was provided. After that, the concentration glucose standard point was being prepared. In this experiment the tube that was used is tube number 3 which the concentration was 100 mg/dl, 0.5 ml glucose standar, 0.5 ml water, and 1 ml of total ml per tube. After it has been prepared, 2 ml of 1% O-toluidine was added to it and well mixed. Then, the tubes were put in a boiling water bath for 10 minutes. Last, measured at absorbance 630 nm.

2.2.4.2. Measuring of blood glucoseThe blood that was drawn from a vein was transferred into a centrifuge tube. After that, the blood sample was centrifuged. The centrifugation process obtained serum. After the centrifugation process was done, the glucose concentration in the provided serum sample of patient 1, 2 or 3 was determined using the O-toluidine method. 0.05 ml of distilled water (blank) or standard glucose (standard) or serum (test) was added in a clean dry test tube, followed by adding 1 ml of O-toluidine reagent. Then, the content of each tube was mixed. The tube opening was covered with aluminium foil. After the tube opening has been covered, the tubes were put in a boiling water bath for 10 minutes. Next, test tubes were removed from the water bath and cool under tap water. Last, the absorbance was read at Imax 630 nm and the concentration of Blood Sugar Level (BSL) in the provided blood samples was calculated using the absorbance reading of standard glucose.Chapter 3RESULTS

3.1. Bradford TestIn this experiment, the standard protein which is Bovine Serum Albumin (BSA) was used to be the compound for comparing the binding of it to a dye with the binding of sample protein to a dye. There were five different concentration of BSA that used in this experiment (one group made one protein standards). After protein standards were made, we got the protein standards absorbance which is shown in the table below:

Absorbance at 595 nmBSA concentration (mg/mL)

12Average

XY

0,1720,1730,17250,125

0,280,2780,2790,25

0,4890,490,48950,5

0,6390,6460,64250,75

0,8920,890,8911

Table 1. Data for absorbances measurement and protein standard (Bovine Serum Albumin) concentration.

Then, we made the standard curve based on the protein standards absorbance which is shown in the figure below:

Figure 7. Standard Curve for Bradford TestFrom the above standard curve, we got the formula for measuring the concentration of protein in our samples which is y = 1,2447x 0,091. The meaning of the statement R2= 0,99531 is the linear shape was valid or can be used. The data of our samples absorbance and the calculation of the protein concentration in our samples is shown in the above table:

Sample12Mean (x)*

I0.5040.5110.5075

II1.2351.2471.241

NOTE: Sample I : Tiara Sample II : AdiTable 2. Data of absorbances measurement of the samples.

The calculation of protein concentration is:Formula y = 1,2447x 0.091 ; *( x = mean of the absorbance)

Tiaras sample:Adis sample:y= 1,2447(0.5075) - 0,091y = 1,2447(1.241) - 0,091 = 0.63133-0.091 = 1.543804-0.091 = 0.54033 = 1.452804 Because we have diluted the sample with the comparison of 1:100, so the resulted concentration above must be multiplied by the dilution factor (100) to get the real concentration of protein in serum.So, Tiaras protein concentration =Adis protein concentration = y= 0.54033 x 100 y= 1.452804 x 100 = 54 mg/ml = 145.28 mg/ml3.2. Protein Separation Through SDS-PAGE

Figure 7. The gelNOTE: Es Adis Ep Adis M DNA marker Es Tiaras Ep Tiaras

The image above shown that well 1 was loaded with Ep from Tiaras sample, well 4 was loaded with Es from Tiaras sample, well 5 was loaded with DNA marker, well 6 was loaded with Ep from Adis sample, and well 8 was loaded with Es from Adis sample. The length of the gel (L) was 4 cm, and the distance between the first line/top part and the last line/bottom part (d) of the chosen band was 2.1 cm.To measured the standard curve, the DNA marker have to be visible in order to know the size of the band. But in our gel result, the DNA marker was not shown. So we had to use DNA marker from other group (B2-4) for measuring the standard curve, which is shown in the figure of the gel result above:

The above figure shows that the DNA marker that was shown up were 75, 50, 37, and 25 kdalton. The length of the maximum protein running in the gel (L) was 4 cm. The distance for each marker band is shown in the below table:Size (dalton)d (cm)

75.0000.7

50.0001

25.0002.1

Table 3. Data of specific sizes distances After that, we have to know the data of the axis y = log of size of the band and the axis x = Rf (Retention factor) which can be got from the formula: d/L (distance per length). The result of the calculation and the data that we got is shown in the below table:Size (dalton)Log of Size (y)d (cm)Rf (x)

75.0004,880.70,175

50.0004,7010,25

25.0004,402.10,525

Length= 4 cmTable 4. Data for axis y (log of size) and x (Rf).

Then, we made the standard curve for SDS-PAGE based on log of size and Rf that we got from the previous step. The standard curve is shown in the above figure:

Figure 10. Standard Curve for SDS-PageFrom the above standard curve, we got the formula for measuring the molecular mass of the protein in bands in SDS-PAGE, which is y = -1.2907x + 5.066. Before we calculate the molecular mass of the protein, we have to know the Rf (Retention factor) first. The band that we used was the band with the 2.1 cm of the distance (d = 2.1 cm). So the Rf from that band is : Rf (x) = d/L = 2.1/4 = 0.525And the calculation of the molecular mass of the protein in band is:y = -1.2907x + 5.066 = -1.2907 (0.525) + 5.066 = - 0.684071 + 5.066 = 4.381929 (log of size)The calculation above resulting the size which is log-1 4.381929If we antilog the size resulting the different value of the size which is 23,988.33.So it can be concluded that the molecular mass for the protein band is 23,988.33 dalton.

3.3. Enzyme-Linked Immunosorbent Assay

StandardAbsorbance (nm)Concentration(ng/ml)

10,0410

20,0865

30,15620

40,38850

50,8150

Table 5. Data of Standard set AFP

Figure11. Standard Curve for ELISA test.From the standard curve in the above figure, we got the formula for measuring the antigen AFP concentration. The formula is y = 196.2x 12.72 ( y protein concentration ; x absorbance ).To calculate the antigen AFP concentration using the formula, the absorbance of the sample must be known. The absorbance of each sample is shown in the below table:SampleAbsorbance (nm)

10.020

20.033

The calculation of antigen AFP concentration in serum are:Sample 1: y = 196.2x 12.72 = 196.2(0.020)-12.72 = |-8.796| = 8.80 ng/mlSample 2: y = 196.2x 12.72 = 196.2(0.033)-12.72 = |-6.2454| = 6.25 ng/mlSo it can be concluded that the antigen AFP concentration in sample 1 is 8.80 ng/ml and the antigen AFP concentration in sample 2 is 6.25 ng/ml.

3.4. Colorimetric Determination Of Blood Sugar LevelThe glucose standard was used to determine the standard glucose absorbance. The different concentration of glucose standard were used by each group (our group used 100 mg/dl), so it resulting the variety of standard absorbance. The standard glucose absorbance and its concentration are shown in the below table:

StandardResult 1Result 2Mean AbsorbanceConcentration

10.2140.2160,21560

20.3310.3310,33180

30.3460.3470,347100

40.4560.4580,457120

Table 6. Data for standard glucose absorbance and its concentrationThen we made the standard curve of the standard glucose solution based on the data of the above table which is shown in the below figure:

Figure 12. Standard curve of Standard Glucose Solutions.From the above standard curve figure, we got the formula for measuring the blood sugar level in the samples. The formula is y = 252.22x + 4.8764. The meaning of the statement R2= 0,99531 is the linear shape was valid or can be used. To calculate the blood sugar level in the samples, the absorbance (x) of each sample also have to be known. The absorbance of each sample is shown in the below table:SampleAbsorbance (nm)

10.2625

20.3625

The calculation of blood sugar level in the serum samples are:Tiaras sample: y = 252.22x + 4.8764 y = 252.22 (0.2625) + 4.8764 = 71.08 mg/dlAdis sample: y = 252.22x + 4.8764 y = 252.22 (0.3265) + 4.8764 = 87.23 mg/dlSo it can be concluded that the blood sugar level in Tiaras serum is 71.08 mg/dl and the blood sugar level in Adis serum is 87.23 mg/dl.

Chapter 4DISCUSSION

The first experiment that was done in this proteomic session was the Bradford test. The Bradford test is some method to determining the total serum protein that is mostly consist of albumin and globulin. Total serum protein is the total amount of protein that is found in blood. This experiment was also done to determine the appropriate dilution factor of the solution. The process in this experiment was depend on quantitating the binding of dye to an unknown protein and comparing this binding to that different amount of a standard protein. The standard protein that was used in this experiment is the Bovine Serum Albumin. This standard have to be diluted in order to adjust the protein concentration, so that it falls in standard range. To measuring the protein concentration in the samples, the absorbance of the standards and samples have to be known. So the spectrophotometer was used at 595 nm to measured the absorbance of them. The spectrophotometer that was used in this experiment have to be set at 595 nm because the absorbance is in the optimum point for the form of binding dye to protein. Optimum means the absorbance was at the peak and cannot go up any further. After the absorbance of protein standards and samples was determined, we got the formula for measuring the protein concentration in samples serum protein by made a standard curve. The formula that we got for measuring the protein concentration was y = 1,2447x 0,091. From the calculation of protein concentration using the formula that we got from the standard curve and multipled it with 100 (because the experiment required 100 times of dilution), we got different result of the total serum protein in each sample. Tiaras total serum protein concentration is 54 mg/ml and Adis total serum protein concentration is 145.28 mg/ml. These results showed the abnormal amount of total serum protein concentration in both samples. Because the normal total serum proteins in healthy adults are 55 90 mg/ml. So the results of both samples were said to be abnormal because in Tiaras sample the concentration was below the normal range, on the other hand Adis sample concentration was above the normal range. The abnormal results that can be known by comparing the results with the normal range of healthy adults might be happened because of the human error in the process of mixing materials, pipetting the volume of unknown sample or dye reagent, and other possible errors. Because a deficiency (< 20 l) or excessive (> 20 l ) amount of the samples or dye reagent could cause an increased and decreased in number of total serum. The abnormal results also can be happened because the total protein in the samples were low.

Because of many proteins that have difference in size and shapehave nearly identical charge and mass ratios, the second experiment in this proteomic session which is the protein separation through SDS-PAGE can be used to determine the molecular mass of protein in the total serum protein of the samples. This experiment can determined the molecular mass of protein in the total serum protein because this experiment was used the electrophoresis process which is the process of separating macromolecules in an electric field. SDS-PAGE was the method that used in this experiment. This method used a discontinuous polyacrylamide gel as a support medium and sodium dodecyl sulfate (SDS) to denature the proteins. The electrophoresis separation of proteins performed in polyacrylamide gels because it restrain larger molecules from migrating as fast as smaller molecules. The protein migration in the electrophoresis was start from the katode (-) to anode (+). It is because protein has (-) charge, the SDS give negative charge and denaturate the protein. The protein migration is based on their mass. After doing several processes, we got the image of our gel which is the well 1 was loaded with Ep from Tiaras sample, well 4 was loaded with Es from Tiaras sample, well 5 was loaded with DNA marker, well 6 was loaded with Ep from Adis sample, and well 8 was loaded with Es from Adis sample. The length of the gel (L) was 4 cm, and the distance between the first line/top part and the last line/bottom part (d) of the chosen band was 2.1 cm. The Ep fraction in the image have to be more thick than the Es, because it precipitated with ethanol. The thick protein that appears on the gel is Albumin, which normally have the mass of 67.000 dalton. To measured the standard curve, the DNA marker have to be visible in order to know the size of the band. But in our gel result, the DNA marker was not shown. So we had to use DNA marker from other group (B2-4) that shown the size of the chosen band was 25.000 dalton. After that, we have to know the data of the axis y = log of size of the band and the axis x = Rf (Retention factor) which can be got from the formula: d/L (distance per length).Then, we made the standard curve for SDS-PAGE based on log of size and Rf that we got from the previous step. From the standard curve, we got the formula for measuring the molecular mass of the protein in bands in SDS-PAGE, which is y = -1.2907x + 5.066. Before we calculate the molecular mass of the protein, we have to know the Rf (Retention factor) first. The band that we used was the band with the 2.1 cm of the distance (d = 2.1 cm). So the Rf from that band is : Rf (x) = d/L = 2.1/4 = 0.525And in the end, we got the amount of molecular mass of the protein in band which is 4.381929 (log of size). Because the size is log-1 4.381929, we have to antilog it and resulting in the different amount of molecular mass of the protein band is 23,988.33 dalton.The third experiment that was done is the Enzyme-Linked Immunosorbent Assay (ELISA) which is done to get the quantitative determination of the antigen AFP cocentration in human serum. On this experiment, there was an antibody-antigen binding detection based on enzymatic reaction. AFP (Alpha-fetoprotein) is a protein that encoded by the AFP gene in human body. AFP has no known function in healthy adults, so the level of it in healthy adults are low. AFP used as a biomarker to detect a subset of tumor. From the experiment that was done by MRIN assistance, the concentration of antigen AFP in the samples serum are 8.80 ng/ml in sample 1 and 6.25 ng/ml in sample 2. These results show that both of our samples were healthy or normal because the results are falls in the normal range (normal = < 20 ng/ml) of healthy person.The last experiment that was done in this proteomis session is Colorimetric determination of blood sugar. Blood sugar is a term used to refer to the level of glucose in blood. Glucose concentration in blood was effected by diet and hormones. Glucose is the most important carbohydrate fuel in the body that the majority of this circulating glucose comes from the diet in the fed state. In this experiment the blood glucose level was measured from serum sample. We used the colorimetric in measuring the blood glucose and used the O-toluidine method to determine or measuring the BSL by forming blue-green complex when it was boiled (with acetic glacial acid).In the experiment the glucose standard was used to determine the standard glucose absorbance. The different concentration of glucose standard were used by each group (our group used 100 mg/dl), so it resulting the variety of standard absorbance. And the standard curve was made after that.From the standard curve, we got the formula for measuring the blood sugar level in the samples. The formula is y = 252.22x + 4.8764. The meaning of the statement R2= 0,99531 is the linear shape was valid or can be used. To calculate the blood sugar level in the samples, the absorbance (x) of each sample also have to be known. The absorbances that we got are Tiaras is 0.2625 and Adis is 0.3625.In the end we got the result which is the blood sugar level in Tiaras serum is 71.08 mg/dl and the blood sugar level in Adis serum is 87.23 mg/dl.After doing these whole experiments in the proteomic session it concludes that the protein concentration of humans blood can be determined by using several compounds that responsible for the separating process of protein molecule with other molecules. It also conclude that the molecular mass of protein can be determined. To know the absolut amount of protein concentration or mass, we have to be careful in doing the processes. Because an error in process can makes the result wrong. If the result wrong we can be wrong in examine the persons condition, because if the result showed the abnormal amount of protein from persons sample, it indicates to an abnormal health and indicate to diseases that related to the protein. Whereas, the abnormal amount of the result might be the result of the human error in doing the processes.

REFERENCES

1. Anonymous. Laboratory Protocol For Fundamental Medical Science 1.Tangerang: Faculty of Medicine-Universitas Pelita Harapan. Mochtar Riady Institute For Nanotechnology. 2010.2. http://www.medterms.com/script/main/art.asp?articlekey=65543. http://themedicalbiochemistrypage.org/protein-structure.html4. http://ww2.chemistry.gatech.edu5. http://www.ehow.com/about_5502288_blood-plasma-regulations.html6. http://www.mcb.uct.ac.za/sdspage.htm7. http://en.wikipedia.org/wiki/Protein_precipitation8. http://en.wikipedia.org/wiki/Protein_precipitation9. http://homepages.gac.edu/~cellab/appds/appd-g.html