Proteomic Report

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Transcript of Proteomic Report

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 produc