For this experiment, a software named Gel Analyzer was used in order to obtain the molecular weight of the unknown proteins with the help of a protein ladder with known molecular weight and protein concentration. The software also generated a curve with a coefficient determination (r2) of the fit which is equal to 0.998. An equation was also generated from the software to be able to determine the molecular weight of the unknown proteins. Results obtained show that the proteins of the cellular fractions of the chicken liver
The Figure 2 shows the examples of various amplification results for the tested two pairs of primers. The seven isolates tested, i.e. A. hydrophila ATCC 7699, SfB, SfN, SfL, SfM, SfP, and PfKT9 had 685 bp amplicon, however when were amplified using the second primers, only the control isolate, SfB, and PfKT9 had 760 bp amplicon. The isolate SfN had two amplicons, which were different from the control isolate; isolate SfL and SfP had single amplicon, but was different from the control, while the isolate SfM did not have
1.1 Abstract The purpose of quantitative analysis of protein using a spectrophotometer is to measure the concentration of proteins in a given sample. The experiment is conducted by laboratory method (Biuret Test) and using spectrophotometer to analyze the absorbance of reactants at 540 nm, hence determining the concentration of the proteins in a given sample. The purpose of stopped enzyme assay to study B-galactosidase is to determine the effect of temperature and concentrations of substrate on enzyme activity. B-galactosidase breaks down the disaccharide lactose into simple sugars glucose and galactose. However, glucose is a colorless compound hence it has to be substituted with a compound that is detectable by a visible color change.
Figure 7: Biosynthesis of Phospholipids Sphingosine Sphingosine is an amino alcohol that contains a long, unsaturated hydrocarbon chain. In sphingomyelin and glycolipids, the amino group of sphingosine is linked to FAs by an amid bond. In sphingomyelin the primary hydroxyl group of sphingosine is esterified to phosphoryl choline. In glycolipids, the sugar component is attached to this group. The simplest glycolipid is cerebroside, in which there is only one sugar residue, either Glc or Gal.
There is a non-covalent interaction between four chains. The four heme groups provide four binding sites for oxygen (Marengo; 2006) As described by Max Perutz in 1959, the three dimensional structure of hemoglobin is similar to myoglobin but oxygen affinity in hemoglobin is more than that of myoglobin. Figure 1: Structure of Hemoglobin (http://www.infobiochem.com/2014/12/Hemoglobin-and-its-defects.html) Synthesis: This complex protein molecule is synthesized in series of steps, it begins with synthesis of d-aminolevulinic acid (ALA) and porphobilinogen and then modification of terapyroll ring. The synthesis of heme part involves enzymes of mitochondria and
SOD activity was calculated in terms of units/mg protein. Total protein thiols (TPT) - This assay is based on the principle of formation of relatively stable yellow color by sulphydryl groups with DTNB (Moron et al., 1979). Briefly, 0.2 ml of liver homogenate was mixed with phosphate buffer (pH 8.0), 40 µl of 10 mM DTNB and 3.16 ml of methanol. This mixture was incubated for 10 min and the absorbance was measured at 412 nm against appropriate blanks. The total thiol content was calculated by using ε=13.6x10 cm-1 M-1 Sedlak & Lindsy, 1978).
To 0.05 ml of carbonate buffer (0.05 M, pH 10.2) and 0.5 ml of EDTA (0.49 M) was added. The reaction was initiated by the addition of 0.4ml of epinephrine and the change in optical density/min was measured at 480nm. SOD activity was expressed as units/mg protein change in optical density/min. 50% inhibition of epinephrine to adrenochrome transition by enzyme is taken the enzyme unit. Calibration curve was prepared by using 10 -125 units of
There are currently known that over 170 amino acids occur in organisms but only 20 are commonly found in proteins. R groups determine the chemical properties of the amino acids. The simplest amino acids have hydrocarbons as side chains which are neutral and non-polar or hydrophobic. They are not soluble in water. Amino acids with a polar R group are neutral and polar or hydrophilic.
The volume in the test tube was adjusted to 0.1 ml with appropriate buffer. Five milliliters of protein reagent was added to the test tube and the contents were mixed either by inversion or vortexing. The absorbance at 595 nm was measured after 2 min and before 1 hr in 3 ml cuvettes against a reagent blank prepared from 0.1 ml of the appropriate buffer and 5 ml of protein reagent. The weight of protein was plotted against the corresponding absorbance resulting in a standard curve used to determine the protein in unknown
Wang et al performed SMFS on a shallow trefoil knot protein, bovine carbonic anhydrase B, and stretched it to a tightened knot. (110) A figure-eightknotted protein, phytochrome, was stretched by Bornschlogl et al using SMFS based on atomic force microscopy (AFM) as well as SMD simulations. (106) The unfolding force of phytochrome was determined to be ~ 70 pN, which is not as high as many mechanically stable proteins such as I27 domain of human titin (~200 pN). In addition, both their experimental and simulation results revealed that the tightened figure-eight knot contains 17 to 19 amino acid residues as shown in Figure 1.11. Further discussion about the tightening of the knot can be found in chapter
Since the current run from negative (top) to positive (bottom), the proteins move toward the bottom. When the electricity is turned on, the proteins and Tris-glycine enter the stacking gel. In stacking gel with pH 6.8, the N-terminal amino group of the proteins and amino acids are protonated at equilibrium, which makes them less negative. The average electrophoretic mobility is very slow. A Gly-chloride ion boundary is formed since glycine moves slower than chloride ion.
Part C: Change the amount of the substrate First, the blank was prepared according to table 2 without the enzyme addition. The enzyme was added later after the blank was measured by the spectrophotometer. Table 2: The amount of Sodium Phosphate Buffer pH 7.0, L-Dopa, and enzyme needed in each cuvette. Cuvette 1 Cuvette 2 Cuvette 3 Cuvette 4 Cuvette 5 Sodium Phosphate Buffer PH 7.0 (mL) 2.40 2.20 1.80 1.60 1.10 L- Dopa (mL) 0.20 0.40 0.80 1.00 1.50 Enzyme (mL) 0.40 0.40 0.40 0.40 0.40 For example, to prepare the cuvette 1, 2.40 mL of buffer pH 7.0 was measured by the micropipette P-1000, and was added into cuvette labeled #1 for the second set of cuvette. Next, 0.20 mL of L-dopa was measured by the micropipette P-1000 and was added into