Quiz Questions On The Properties Of A Protein At Dna Level

This experiment will be done as the first step in determining an unknown protein. The second part of the protein determination will be done using the Western blot technique. We will use the Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) method to separate proteins by their molecular weight. This method is often used to determine the purity and weight of a protein. Electrophoresis is a method of separating proteins based on their chemical and physical properties. Gel electrophoresis has a gel support mediums that includes polyacrylamide, starch and agarose. The gel is chemically inert, it is easy to handle, and can me made to fit a desirable porosity. This makes it good to use for molecules with high molecular weights. After separating the gel, it will be stained with Coomassie Blue to visualize the protein.

Having removed the detergent, the protein will refold. As shown by the Anfinsen experiment the polypeptide sequence determines the folding and therefore the three dimensional structure. As the polypeptide sequence is unaltered refolding can occur through the process of nucleation aggregation and compaction. In order to test that the protein was no longer denatured, the absorbency of the solution at 412nm could be measured and compared with the graph in figure 1 above, it should match the plot of standard ovalbumin in the absence of SDS.

Proteins are polymeric chains that are built from monomers called amino acids. All structural and functional properties of proteins derive from the chemical properties of the polypeptide chain. There are four levels of protein structural organization: primary, secondary, tertiary, and quaternary. Primary structure is defined as the linear sequence of amino acids in a polypeptide chain. The secondary structure refers to certain regular geometric figures of the chain. Tertiary structure results from long-range contacts within the chain. The quaternary structure is the organization of protein subunits, or two or more independent polypeptide chains.

Next, the biuret test for protein turned solutions in test tube 9 and 12 purple, the biuret test was positive, which proves that protein was present in the solutions, whereas test tubes 1,2,3,4,5,6,7,8,10, and 11 had negative results, this is because those solutions do not have peptide bonds and a peptide bond is necessary for the biuret test to be positive. An unknown was also tested in this experiment and we have concluded that the unknown #267 is a protein because it

The purpose is to transfer biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane. An antibody is used to specifically detect its antigen for protein analysis. The specificity of the antibody-antigen interaction enables a target protein to be identified in the middle of a complex protein mixture.

The purpose for this experiment is to determine if different proteins denature, physically change, at the same temperature. By doing this, we can prove that not all proteins are made the same, and that they have different structures.

Western blot is used to separate and identify proteins by taking mixtures of proteins and separating them based on molecular

Campbell and Farrell define proteins as polymers of amino acids that have been covalently joined through peptide bonds to form amino acid chains (61). A short amino acid chain comprising of thirty amino acids forms a peptide, and a longer chain of amino acids forms a polypeptide or a protein. Each of the amino acids making up a protein, has a fundamental design that comprises of a central carbon or alpha carbon that is bonded to a hydrogen element, an amino grouping, a carboxyl grouping, and a unique side chain or the R-group (Campbell and Farrell 61).

Proteins are macromolecular polymers, built up of chains of repeating units called amino acids. There are twenty different naturally occurring amino acids, all of which have the vital amino (-NH₂) and carboxylic acid (-COOH) groups bonded to the central carbon atom, as well as an “R” group specific to that amino acid. Proteins are versatile and useful molecules, they can be used in many different roles in the body. For example, the catalysis of biological reactions, the transmission of messages around the body and the transportation of molecules (eg. the transportation of oxygen by haemoglobin). Proteins are coded for by the sequence of nucleotide bases in the DNA sequence of genes specific to the protein molecule in question. This sequence of bases is copied in the process of transcription which takes place in the nucleus of the cell. The copied sequence then undergoes post-transcriptional modification. This allows the introns (which are of no use in translation) to be removed from the sequence and the exons to be organised into the correct sequence to code for a specific protein. This modified sequence is then used in the process of translation to code for the amino acid sequence of the protein. Translation takes place in the ribosomes of the cell and is the process which results in the final formation of the linear polypeptide chain of the protein. This polypeptide chain is the basic primary structure of the protein. Proteins have four basic levels of

ProtParam was used to determine the physiochemical properties of the protein sequence. The results of primary structure analysis show that DT-STXB fusion protein is hydrophilic in nature due to the large number of polar residues. Also, the primary structure analysis suggests that the average molecular weight of DT-STXB is considered to be 53.64 kDa. Isoelectric point (pI) of a protein is defined as the pH at whichthe net charge of the surface of the protein is zero. At pI, the proteins are stable and dense. The calculated pI value of DT-STXB is 5.11 (pI <7). Although Expasy’s ProtParam computes the the extinction coefficient for a range of (276, 278, 279, 280 and 282 nm)wavelength, extinction constant of DT-STXB at 280 nm is 49850 M–1 cm–1 with respect to the low concentration of Cys, Trp and Tyr, indicates that this fusion protein cannot be analyzed using UV spectral methods. The computed protein concentration and extinction coefficients are useful in quantitative study of protein–protein and protein–ligand interactions in the solution.

Proteins can be identified through western blot analysis. The procedure separates proteins by electrophoresis and transferred onto nitrocellulose sheets on which the protein is immobilized. Protein bands can be identified on the sheets by binding to their labeled antibodies and compared to a molecular marker to establish weight (kD).

Over the past few decades, the scientific community’s knowledge of protein structure has increased exponentially. Through the use of NMR and, primarily, X-ray crystallography, valuable information pertaining to the three-dimensional structure of proteins and enzymes alike have been found and recorded. Understanding protein structures and crystallography methods plays a major role in purifying drugs as well as pharmaceutical design in order to efficiently deliver drugs in to the body. (Durbin, Feher, 1996) The formation of viable protein crystals is necessary for crystallography. Varying

Incorporation of air bubbles and absorption of protein molecules to the air- liquid interface also cause denaturation of proteins. The extent of change in conformation of proteins is based on the flexibility of the proteins. Highly flexible proteins denature more readily than the rigid ones do at the air- liquid interface.

Proteins can also be fractionally precipitated by adjusting the pH, temperature and dielectric constant. Variations in pH also change the state of ionization of the functional groups, and hence, the net charge of the protein. Usually, the protein’s solubility is at its least at the isoelectric point pI and increases on either side of the pI. At the pI, the net charge of the protein is zero and the protein molecules do not repel each other. As a result, protein-protein interactions are increased and the solubility is at its lowest. (Boyer, 2000)

Solubility of the protein is due to the exposure of the hydrophilic group, in glycolation there is attachment of hydrophobic group without effect of the on the structure activity of protein. glycolation involve the increase in the hydrophobicity of the protein. Higher exposure of the hydrophobic group of amino acid and result in structural changes in the amino acid. Another effect of glycolation involve decrease in solubility due to masking of surface hydroxyl group of protein, with apropiat pH adjustment so it come closer to its isoelectric point where net charge becomes