Discussions Bradford reagent is used to determine the concentration of protein in the experiment, it is a red brown acidic solution in the presence of protein. Bovine serum albumin (BSA) used for various biochemical applications include ELISA which is Enzyme-Linked Immunosorbent Assay, high content of screening assays, western blotting and immunohistochemistry. Furthermore, Bovine Serum Albumin can also be used to determine the quantity of other proteins which is by comparing an unknown quantity of protein to known amount of BSA. In this experiment, Bovine serum albumin (BSA) solution is used as protein concentration standard as the stability of bovine serum albumin will increase signal in the assays and lack of effect in biochemical reactions. Both of these solution are used in part 1 and part 2. A graph of absorbance against volume of BSA protein concentration is plotted in part 1. A graph is shown approximately whereby the absorbance increases when the volume of BSA concentration increases. Since the …show more content…
Bradford reagent is light blue in colour, thus the straight line doesn’t start from the origin although protein is not added. The Beer-Lambert Law is most accurate between Abs of 0.05 to 0.70. Above 0.70, the measured Abs tends to underestimate the real Abs. Below 0.02 Abs, many instruments are not accurate enough. The Beer-Lambert law related the attenuation of light to the properties of the material through which the light is traveling (Jim Clark, 2007). The equation of the Beer-Lambert law is shown below: Source: (http://chemwiki.ucdavis.edu/Physical_Chemistry/Spectroscopy/Electronic_Spectroscopy/Electronic_Spectroscopy_Basics/The_Beer-Lambert_Law#The_Beer-Lambert_Law) Thus, from the equation above, the greek letter epsilon is called the molar absorptivity, sometimes it is also referred as molar absorption coefficient.
Thus, the larger the molar absorptivity, the more probable the electronic transition. In the UV spectroscopy, the concentration of the sample solution is measured in molL-1 and the length of the light path in cm. Hence, given that absorbance is unitless and the unit of molar absorptivity are L mol-1 cm. Since the units of molar absorptivity is always the above, it is customarily reported without unit. The derivative of the Beer-Lambert law is shown below: Step 1: Assumption one relates the absorbance to concentration and can be expressed as A∝ c (1) Step 2: The absorbance (A) is defined via the incident intensity Io and transmitted intensity I
by A=log10 (Io/I) (2) Step 3: Assumption two can be expressed as A∝ l (3) Step 4: When combining Equations 1 and 3: A∝ cl (4) Step 5: This proportionality can be converted into an equality by including a proportionality constant. A= ϵcl (5) Step 6: This formula is the common form of the Beer-Lambert Law, although it can be also written in terms of intensities: A=log10 (Io/I) = ϵlc
Data from Table 1. confirms the theory that as the concentration of glucose increases so will the absorbance of the solution when examined with the glucose oxidase/horseradish peroxidase assay. Glucose within the context of this assay is determined by the amount of ferricyanide, determined by absornace, which is produced in a one to one ratio.1 Furthermore when examining the glucose standards, a linear calibration curve was able to be produced (shown as Figure 1). Noted the R2 value of the y = 1.808x - 0.0125 trend line is 0.9958, which is statistically considered linear. From this calibration curve the absorbance values of unknowns samples can be compared, and the correlated glucose concentration can then be approximated.
The analyzed yellow#5 wavelength was determined to 395nm because the actual wavelength 427nm was restricted in the Micro lab. The R2 value of the graph is 0.9827, and the level of data accuracy it indicated extremely weak data correlation. The first one dilution data points excluded from the standard curve because the point is not in the linear curve. The first concentration and absorbance value are the highest point in the graph that cannot connect as linear with another data point. After removing the first data point, the standard curve is clear and make
Absorbance was defined as: log I_o/I where I_o is incident light and I is the transmitted light. Fluorescence emission spectrum is different from fluorescence excitation spectrum because it records different wavelengths of chemical s...
Data table 1 Well plate Contents Glucose concentration A 3 drops 5% sucrose + 3 drops distilled water Negative B 3 drops milk+3 drops distilled water Negative C 3 drops 5% sucrose +3 drops lactase Negative D 3 drops milk +3 drops lactase 15+ E 3 drops 20% glucose +3 drops distilled water 110 ++ Questions B. In this exercise, five reactions were performed. Of those reactions, two were negative controls and one was a positive control.
The least absorbed was the 4 Celsius with only 0.040 absorbed. The most absorbed was -20 Celsius with 0.219 absorbed.
One of the most primitive actions known is the consumption of lactose, (milk), from the mother after birth. Mammals have an innate predisposition towards this consumption, as it is their main source of energy. Most mammals lose the ability to digest lactose shortly after their birth. The ability to digest lactose is determined by the presence of an enzyme called lactase, which is found in the lining of the small intestine. An enzyme is a small molecule or group of molecules that act as a catalyst (catalyst being defined as a molecule that binds to the original reactant and lowers the amount of energy needed to break apart the original molecule to obtain energy) in breaking apart the lactose molecule. In mammals, the lactase enzyme is present
The affects of pH, temperature, and salt concentration on the enzyme lactase were all expected to have an effect on enzymatic activity, compared to an untreated 25oC control. The reactions incubated at 37oC were hypothesized to increase the enzymatic activity, because it is normal human body temperature. This hypothesis was supported by the results. The reaction incubated to 60oC was expected to decrease the enzymatic activity, because it is much higher than normal body temperature, however this hypothesis was not supported. When incubated to 0oC, the reaction rate was hypothesized to decrease, and according to the results the hypothesis was supported. Both in low and high pH, the reaction rate was hypothesized to decrease, which was also supported by the results. Lastly, the reaction rate was hypothesized to decrease in a higher salt concentration, which was also supported by the results.
Moreover, the sensitivity and specificity of the western blot (Immunoblotting) enables it a common technique for determining specific protein levels in clinical samples. Since the antibody is specific to the antigen immunospecificity it enables the target protein to be identified. Western blotting can produce quantitative data about that protein, which in this case shows the difference between bands in each of the protein samples. The western blot is an analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. The proteins are then transferred to a membrane (in this case, nitrocellulose), where they are stained with antibodies specific to the target protein [1] [2].
Thyroid and metabolism hormones play a large role in the daily lives of all living species. Thyroid hormones regulate the metabolism and the metabolism is responsible for maintaining a specific range for the biochemical reactions that occur in the body (Martini 2014). The most important hormone for metabolic maintenance is thyroxine (T4). This hormone also plays a large role in body heat regulation. It is produced by the pituitary gland and secreted by the thyroid gland. The thyroid releasing hormone (TRH) must trigger the thyroid stimulating hormones (TSH) to release thyroid hormones to the thyroid gland. These hormones are under control of the hypothalamus, or main neural control center. Propylthiouracil (PTU) is a medication used to treat
...eases, including temperature. It is determined from the data that the reaction is more likely to have a step wise mechanism than a concerted due to the small – ΔS and a relatively large value of ΔH from the tables. Due to some errors, it is best to perform another experiment for future protocols. In addition with the variance the 35°C where at one point the absorbance levels off and then increases. In comparison to the rate constant against temperatures, at 25°C it is higher than 35 and 45. More test is required to ensure proper determination of the rate constant at those temperatures.
Mader, S. S. (2010). Metabolism: Energy and Enzymes. In K. G. Lyle-Ippolito, & A. T. Storfer (Ed.), Inquiry into life (13th ed., pp. 105-107). Princeton, N.J: McGraw Hill.
Figure 3: The absorbance of peroxidase reactions over two minutes using pH 3, pH 5, pH 7, and pH 9.
The independent variable for this experiment is the enzyme concentration, and the range chosen is from 1% to 5% with the measurements of 1, 2, 4, and 5%. The dependant variable to be measured is the absorbance of the absorbance of the solution within a colorimeter, Equipments: Iodine solution: used to test for present of starch - Amylase solution - 1% starch solution - 1 pipette - 3 syringes - 8 test tubes – Stop clock - Water bath at 37oc - Distilled water- colorimeter Method: = == ==
...le that has an intensity of 10-3 W/m2 can be determined2: db = 10 log 10 ( 10-3/10-12 ) = 90-dB.
= Before conducting the experiment I would conduct a simple test for the protein by placing a sample of the albumen into a test tube and add biurett reagent. This contains copper (II) sulphate and sodium hydroxide.