Gapdh Lab Report

The enzyme lactate dehydrogenase (LDH) catalyzes the last step of anaerobic glycolysis that is important for the normal function of the body. Purification of LDH is essential to understand its structure and function. The purpose of this experiment was to extract and purify LDH enzyme from chicken muscle tissue using a variety of various. Analytical methods such as activity and protein assay were employed to determine the presence and purity of LDH. The cells were initially disrupted and proteins were solubilized. LDH was purified from the ammonium sulfate precipitated protein mixture by affinity chromatography and its activity was studied by

The method used in this experiment is the non- organic extraction where cell lysis solutions, proteinase K, isopropanol and ethanol was used. The functions of these solutions will be further discussed in the discussion.

Protein purification is a process that can be employed to separate a single protein from a larger starting material which may be anything from an organ to a cell. Isolating a purified protein from a larger fraction enables further analysis such as determination of amino acid sequence, potential biological function, and even evolutionary relationship. (Cuatrecasas 1970) In this experiment, the enzyme lactate dehydrogenase will be purified, this enzyme is found extensively in human cells and catalyzes the conversion of lactate to pyruvate, an essential part in energy production. LDH is a key part of anaerobic energy production especially within glycolysis in which LDH catalyzes the conversion of the reverse reaction, pyruvate to lactate, generating NAD+ from NADH, reproducing the oxidized form of the coenzyme which can be used for oxidative respiration. (Markert 1963) Due to the fact that number of purification steps correlates with the purity of the protein multiple purification techniques will be used to isolate a pure form of LDH. LDH will be isolated from a larger “cytosol” fraction collected from a homogenized rat liver in a previous fractionation exercise. Of the procedures that will be used to isolate and purify proteins from a larger fractionate are a set of techniques collectively known as chromatography. These techniques all have the same premise, in that they consist of a stationary phase, also known as the

Eisenthal, R. & M.J. Danson (Eds.) (2002). _Enzyme Assays: A Practical Approach_. United Kingdom: Oxford University Press

The purpose of this lab was to test if yeast could or could not metabolize different types of sugars. The lab can also display how the different types of sugars affect the rate of respiration in yeast. The yeast was tested with each individual sugar to determine the rate of respiration. The smallest sugar had the highest rate of respiration and the largest sugar had the lowest rate of respiration.

The purpose of this investigation is to test the effect of different sugar sources on yeast respiration.

The yeast Saccharomyces cerevisiae was used for determining the fermentation of various sugars because it is convenient to use and ferments quickly. Throughout the experiment, the only factor that wasn’t constant was the sugars used in the fermentation process. Based on this, it was hypothesized that there would be differences in yeast fermentation of sugars.

Calculations were prepared in advanced for this portion of lab, when obtaining values for protein concentration and volume. Protein samples from the previous lab were removed from the ridge and allowed to thaw, then placed on ice to maintain cool temperature. Two square trays at our lab were labeled with our lab group information. Two empty t1.5 mL tubes were labeled NT and LPS and prepared by adding the 4x protein volume (μg) and 4x RIPA volume (μg) calculated from the previous lab. 40 μL of 2X loading buffer was added to each tube. The ladder was prepared by adding 10 μL of ladder to a 1.5 mL tube with 17 μL of RIPA and

Figure 1 contains gel electrophoresis for protein samples. The lanes were labeled from 1 to 10 from the right to the left. Lane 1 contained the ladder fragment. Lane 2 contained the filtrate. Lane 3 contained the S1 sample. Lane 4 contained the P1 sample. Lane 5 contained the P1 medium salt sample. Lane 6 contained the P1 high salt sample. Lane 7 contained the S2 sample. Lane 8 contained the P2 sample. Lane 9 contained the P2 medium salt sample. Lane 10 contained the P2 high salt sample.

Green fluorescent protein (GFP) comes from the jellyfish Aequorea Victoria is rare proteins with high fluoresce and absorbance. The purpose this experiments is to purify and express a His2-tagged recombinant from of GFP (rGFP) from the E. coli strain BL21(DE3)< pRSETA-GFPUV > through a series of experiments by using Ni+2 agarose affinity chromatography technology. The GFPuv gene (UV-optimized GFP) was over expressed in the E. Coil strain BL21 (DE3) (pLysS) as an n-terminal His6/Xpress epitope tagged bind protein. Then using Ni2+ Agarose affinity chromatography to obtain purification of the crude extract. Then observe under the long wavelength UV light, the activity of the rGFP in the column fraction. Bradford assay was performed to obtain the total protein amount. When calculating the

The GLAT enzyme itself belongs to the histidine triad super family and is a member of branch III. This enzyme shows specific nucleoside monophosphate activity and is a homodimer with each monomer containing a single domain comprised of 6 α- helices and a β-sheet which is formed by 13 antiparallel and 1 parallel strand. The mechanism of this enzyme is described as having ping-pong kinetics with the following two steps. In the first step the active site histidine attacks R-phosphorus of UDP-glucose which displaces glucose-1-phosphate and forms a covalent intermediate. The second step involves the previously formed intermediate reacting with galactose-1-phosphate to displace the histidine and produce UDP-galactose. (Facchiano, 103-104).

In this experiment, a culture of yeast with trpl-289 allele was obtained. Four Eppendorf tubes were labeled 10-1, 10-2, 10-3, and 10-4. Using a pipette, 900 μl of sterile water

Gelatin is observed to have a lower absorbance reading than lysozyme in only the Bradford assay, while in the BCA assay gelatin was observed to have about the same absorbance as lysozyme. In Bradford assay, the color yield for proteins with higher content of tryptophan, tyrosine, or cysteine residue, will be higher. The observed protein sensitivities did not correlates with the amino acid content in the Bradford assay. As presented in Table I, the mole fractions of lysine and arginine in BSA were 10 mole percent and 4 mole percent, respectively. The sensitivity should have been higher in BSA than in the Bradford assy. However, the observed sensitivities did correlates with the amino acid contents in the BCA assay.

Final lab day four; the test media were collected for explanation and interpretation, the results are as listed:

Yeast can reproduce both asexually and sexually, which makes it very easy to grow in the laboratory, as it is very small in size. Mutant yeast can be easily isolated considering yeast consists of a single cell and can be grown as a haploid or diploid. Diploid cells are formed by the combination of MATa and MAT alpha cells. However, under conditions of carbon and nitrogen starvation, the diploid cell will undergo meiosis to produce four haploid microorganisms. Because haploids only have one set of genes, its allele can determine the corresponding phenotype. By mating the mutants, the genetics can be carried out through replica plating with the YPD plates (1). Saccharomyces cerevisiae is one of the most commonly studied strains of yeast, in