Abstract
Affinity purification is a powerful method of isolating a protein of interest from a complex mixture of proteins. the aim of this experiment was to isolate the enzyme GAPDH from a complex mixture of proteins, in this case yeast lysate. in order to achieve this, a range of methods were used such as protein, GAPDH activity and ADH assays, SDS-PAGE and imumunodetection of ADH and GAPDH on western blot. The results showed that the isolation of GAPDH was achieved albeit, extremely with the % recoveries of the elution being abnormally high due to abnormally low initial material values. The results suggested overall that isolation GAPDH from yeast lysate using affinity purification is in fact a somewhat effective method.
Methods
Affinity
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SDS-PAGE Electrophoresis Gel. - Expression of numerous proteins in different fractions of yeast lysate. Marker proteins were utilised in order to identify them under reducing conditions. Polyacrylamide gel was stained with Instant Blue Coomasie Stain which binds itself to proteins allowing band patterns to be viewed. 10ul of molecular weight standards was expelled in the first lane with the remaining fractions in the following lanes. GAPDH rabbit muscle and Yeast ADH was used as a control.
Figure 2. Immunodetection of GAPDH on Western Blot. - Expression of GAPDH in yeast lysate fractions. Presence of GAPDH was assessed using its antibodies. Proteins were transferred from the SDS-PAGE gel to a nitrocellulose membrane for the proteins to be readily bound to the antibodies using an electric current. The membrane was then incubated with the antibodies with the enzyme alkaline phosphate which was used to indicate the presence of GAPDH
Discussion
The aim of the experiment was to isolate GAPDH, from table 2 it
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It was especially high in elution 1 and 2 who had values of 527% and 514% respectively, indicating a high GAPDH activity especially elution 1. This is supported by the fact that elution 1 had the highest specific activity with a value of 0.0017 umoles/min/mg. it is however difficult to see in figure 2, with the number of bands being seemingly lower than there should be.
The SDS-PAGE Electrophoresis (figure 1) contained different samples of the yeast lysate. From the gel the bands are clear and distinct with the YNSMY-YNFT having the thickest and most distinct of columns making the cibracon blue an efficient tool for affinity chromatography. The protein concentration from YNSM to YNW4 fluctuated with a general decreasing trend however the gel does not distinctively show this.
Limitations
The % protein recovery from the ADH assay was 556%, which could be due to a dilution error, most likely that the initial material had abnormally low values. It was expected that elution 3 (YNE3) would have the lowest activity in GAPDH, from table 2 it is clear that it had the highest specific activity but the lowest total activity and thus the lowest % protein recovery.
Overall, the results suggest that, this was somewhat of an effective method in isolation the enzyme GAPDH from yeast
The dark, navy blue colored graph represented the absorbance curve for the S1 sample. The red colored graph represented the absorbance curve for the S2 sample. The green colored graph represented the absorbance curve for the P1 sample. The purple colored graph represented the absorbance curve for the P2 sample. The gaps between the P2 curve was due to the oversaturation that led to the inconclusive spectrophotometer readings. The blue colored graph represented the absorbance curve for the P1 low salt sample. The orange colored graph represented the absorbance curve for the P2 low salt sample. The light blue colored graph represented the absorbance curve for the P1 medium salt sample. The light pink colored graph represented the absorbance curve for the P2 medium salt sample. The light green colored graph represented the absorbance curve for the P1 high salt sample. The light purple colored graph represented the absorbance curve for the P2 high salt
Eisenthal, R. & M.J. Danson (Eds.) (2002). _Enzyme Assays: A Practical Approach_. United Kingdom: Oxford University Press
In this experiment, 4 grams of peeled turnip was used to prepare the enzyme extract opposed to the 1 gram of turnip suggested by Fundamentals of Life Science. Along with the change to the amount of turnip used, the amount of 0.1M phosphate buffer used to prepare the enzyme extract was changed from 50mL to 30mL. The affect of temperature on enzyme activity was not
From the stock substrate solution of 2.5 mM, each group serially diluted at least one different substrate concentration for a total of four different substrate concentrations to be investigated: 1.25 mM, 1.0 mM, 0.75 mM, 0.25 mM. The enzyme concentration was kept constant at 2.0 mM while experimenting on the affect of varying enzyme concentration on the rate and product formation of ONP. Enough 2.0 mM enzyme solution was prepared in the previous part of the project to supply this assay. Using similar procedure to collect absorbance data as the first part, 0.5 mL of 2.0 mM enzyme concentration was placed into the cuvette and used to calibrate the spectrometer at 420 nm. Data was then started, with the immediate addition of 0.5 mL of varying substrate concentrations. Each varying substrate concentration was split between the team and run for a total of 10 minutes, with the exception of the 1.25 mM run. Upon completion, data from each varying substrate concentration was copied to a single Excel sheet and used to produce an absorbance vs. time graph, product formation vs. time graph, Michaelis Menten plot, and Lineweaver-Birk plot. This analysis was used to calculate the V0,Vmax, and Km for β-Galactosidase
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 lab we tried to find what fuels yeast could metabolize and what the yields of the carbon dioxide gas that were produced from the different sugars used. We used 6 different yeast and sugar mixtures. The different yeast and sugar mixtures we used were control, glucose, sucrose, fructose, starch, and saccharin. The results for the 6 different results are presented in Tables 1-6 and Graph 1. Graph 1 is a graph of all the information in Tables 1-6. Each Table and graph is labeled approximately.
10 microliters of the sample is then added and the assay absorption is measured at 340nm. If absorbance was above 1.5, samples were diluted.
After the substrate solution was added, five drops of the enzyme were quickly placed in tubes 3, 4 and 5. There were no drops of enzyme added in tubes 1 and 2 and in tube 6 ten drops were added. Once the enzyme solution has been added the tubes were then left to incubate for ten minutes and after five drops of DNSA solution were added to tubes 1 to 6. The tubes were then placed in a hot block at 80-90oC for five minutes. They were then taken out after the five minute period and using a 5 ml pipette, 5 ml of distilled water were added to the 6 tubes and mixed by inversion. Once everything was complete the 6 tubes were then taken to the Milton Roy Company Spectronic 21 and the absorbance of each tube was tested.
This Lab Report is an analysis of the results of a two-part experiment. In the first part, we used a gel filtration column to separate the components of a mixture composed of protein and non-protein molecules. By doing so we hoped to obtain fractions that contained single components of the mixture, while also gaining insight into the relative molecular weight of each component compared to each other. We would then plot these fractions onto nitrocellulose paper in order to determine which fractions had protein. In the second part, we would use the fractions which we had determined had protein to conduct an SDS-PAGE. By doing so we hoped to determine an estimate on the molecular weight of the proteins present in each fraction by comparing it to a tracker dye composed of a variety of molecules of differing molecular weight.
In our experiment we will use differential centrifugation to isolate the mitochondria of cauliflower and then assay SDH activity using a fixed time assay. We will then measure protein content in our fractions and calculate specific activity and total activity of our fractions.
The purpose of this investigation is to test the effect of different sugar sources on yeast respiration.
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
Conclusion: The yeast was tested multiple times at different concentrations of 100%, 50%, and 10%. As
Restriction Enzyme Digestion – The experiment was begun after putting on gloves to avoid any chemical contact with the skin. Four microtest tubes were obtained, and each of them was labeled to contain the different enzymes or suspect DNA. Two of the microtest tubes were used for suspect one and the two different restriction enzymes, while two other microtest tubes were labeled for suspect two and the two restriction enzymes. After labeling the tubes, the contents that were at the bottom were taken out by slightly tapping them. Then to begin setting up the enzyme reactions, a micropipette was used to obtain 10 μL of the reaction buffer which was added to each of the four test tubes. The buffer is important because it carries the electrical current from the power supply in the gel. After the reaction buffer was in each, the microtest tubes were individually filled with their specific enzymes and DNA, shown in summary through Table 1.1 below. The restriction enzymes are used to cleave the DNA at specific
Different techniques and principles for protein extraction and characterization were demonstrated in this experiment. Various proteins were extracted from different sources: 1.67 g yeast invertase, 1.03 g egg white albumin, and 5.15 g of milk casein. Activity assay for invertase was performed using Benedict’s test and the enzymes inverting action on sucrose was confirmed. Warburg-Christian Method and Bradford Assay were also employed to determine the protein concentration in the albumin and the casein samples. The concentrations for the albumin and casein samples were found to be 0.519 and 0.327 mg/mL, respectively based on Warburg-Christian Assay; and 6.5x10-3¬ and 1.9x10-2 mg/mL