Research on Yeast Fermentation

The preparation for the experiment started by gathering the solutions of enzyme Peroxidase, substrate hydrogen peroxide, the indicator guaiacol and distilled water. Two small spectrometer tubes and three large test tubes with numbered labels. In addition, one test tube rack, one pipet pump and a box of kimwipes were also gathered. Before the experiment, the spectrometer must be set up to use by flipping the power switch to on. Following, the machine was warmed up for 10 minutes and the filter lever was moved to the left. In addition, I set the wavelength to 500 nm with the wavelength control knob. Before the experiment, I had to create the blank solution by pipetting 0.1 ml of guaiacol, 1.0 ml of turnip extract and 8.9 ml water into tube #1. Following the creation of the blank, a control 2% solution was created.

5) 5 mL of sugar was added to the solution and a balloon was placed over the opening of the bottle to minimize the loss of any gas from the system.

We used to florets from the cauliflower and disrupted the cell walls using a cold isolation buffer (0.3 M D-mannitol, 0.02 M phosphate buffer, pH 7.2) and an abrasive, we then strained the homogenate with cheesecloth into a centrifuge tube suppoerted by ice. The homogenate was then centrifuged at 600 g for 10 minutes at 4⁰C. The postnuclear supernatant was removed and centrifuged at 12,000 g for 30 minutes at 4⁰C. The post mitochondrial supernatant was removed and the mitochondrial pellet was resuspended in a cold assay buffer (0.3 M D-mannitol, 0.01 M KCl, 0.005 M MgCl₂, 0.02 M phosphate buffer, pH 7.2). Both samples were stored at

Understanding the activity of enzymes in different muscle types can aid greatly in obtaining more information about other processes such as metabolism of the tissues (Anderson et al., 2012). There are many different methods in order to achieve this information based in two different major categories but the most convenient method is one called continuous assay. This process includes the use of a spectrophotometer to continuously monitor the assay. This method allows for an easy way to calculate the initial rate of reaction as one can establish time points easily. In the lab performed the continuous method was used in order to determine the measurement of the activity enzyme succinate dehydrogenase (SDH). SDH is most commonly found in mitochondria. It is present in many different muscle tissues including the heart, red, and white tissue. In the following lab the enzyme was tested and measured in these three muscle types in the Oncorhynchus mykiss in order to determine which type contained the highest and the lowest activity. This enzyme is involved in multiple processes such as involved in both the Krebs cycle and the electron transport chain (ETC) (Smith, 2014). Its role in

The dialysis tubing will be clamped at one end in order to fill it and then clamped at the other end to seal the filled bag. If the bag is not soft and floppy, the experiment will not work. Blot a bag with a paper towel to absorb the moisture and weigh it, if this blotting process is not done it could interfere with the weight readings creating inaccurate information. After the bags of the solutions are prepared, they will be placed into five different beakers with different solutions. Beakers 1-4 will be filled with tap water and the fifth beaker is filled with 40% sucrose and water. Fill each beaker with just enough water or solution so that the bag is covered and place the bags in the beakers simultaneously and record each time. Every 10 min the bags are to be taken out, blotted, and weighed again before returning them back into their respective beaker for another 10 min. The process is repeated until you have reached 90 min. The weights should be recorded in grams (g).

There were three test tubes in which the experiment was held. A relatively equal sized portion of raw potato (this contained the enzyme [a biological catalyst] hydrogen peroxidase) was placed in each tube. Then, enough water to cover the potato was added. Proceeding this, each of the test tubes were assigned a temperature; cold, room temperature or warm (this was written on the tag so that they were not confused). The test tube destinated ‘cold’ was placed in a ice bath for five minutes. At the same time, the ‘hot’ test tube was placed in a hot water bath for five minutes. Meanwhile, the room temperature test tube sat at room temperature for five minutes. When the five minutes were over, the test tubes were returned to the rack (so that they were able to be observed). Then, the test tubes were allowed to sit at room temperature for five more minutes. Once that period of time was over, 2 ml of hydrogen peroxide (the substrate) was added to each tube.

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.

small wad of absorbent cotton in the bottom of each vial and, using the pipette or syringe, saturate the cotton

The solution is stirred with a stirring rod and stopped as soon as the pink colour disappears.

The data collected of % transmittance can then be used as an indication of changes in membrane permeability of

The absorbance is measured using a Plate reader and a Standard curve is generated. Also, the different types of pipetting techniques are assessed in this Assay.

2. (6 pts) Turn your head to the right. (Create a table* that describes which muscles move which bones across which joints under the control of which nerves)

Incorporation of assay controls included setting up a spectrophotomer and running the chart recorder with a full-scale deflection before the start of the assay. The set recorder had a corresponding value of 1 for the change in the absorbance. Therefore, prior testing was done to observe whether a change occurred in the readings. This helped to indicate that the results were valid, as they could have been affected by a fault during the setting up of the spectrophotometer. On the other hand this was considered as one of the controls for the experiment. Nevertheless, a new cuvette had to be used for each assay.

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

We have timed each of the three experiments for 5 minutes to make sure that our results are correct. Thanks to the program, we got the transmittance and the absorbance, together with the graph portraying our results.