Abstract In this experiment The Bradford Assay, this experiment should prove how much protein and absorbance is in each concentrated solution used. The goal of the experiment is to make sure each data and hypothesis is fulfilled in each experiment conducted. The experiment had two parts to it, part one which had to do with mixing reagents and PBS together and finding how much absorbance was in each cuvette. Part two of the experiment consist of finding how much protein and absorbance is in each product used. The products for part two was: whole mike, muscle milk, and protein milk each of these had different protein and different absorbance. The strategy of the entire experiment was to find the data to plot a slope graph and find the calculations …show more content…
In the part one the experiment the objective was to use measurements in biology and properly measure small volumes using a micropipette. To also learn and understand the application of the spectrophotometry technique. Discover a standard curve by using a known concentration of nitrate. Then finally utilize the standard curve to determine the concentration of the unknown solution. The first step in conducting the experiment is using an indicator brilliant blue dye to add to the concentrated protein solution. The solution will then turn reddish brown because of the high volume in the reagent is unstable. Therefore the reagent binds to the protein in the solution and becomes stable. Using a spectrophotometer, the equipment will measure the protein and absorbance that’s in the solution. To find the measurement of each one must first create a blank cuvette and set the spectrophotometry wavelength to 595nm. Next the student should mix the dye with the reagent and add 1ml of the dye reagent to 3 cuvettes. Then using a 20 µl pipette the student will add 0.250 mg/ml to the 2nd cuvette and followed by the other 3 cuvettes using a new tip each time. After the cuvettes have been filled, para film should be used to cover the solution for at least 5minutes no more than 60mintues. Once they have set for 5 minutes maximum the spectrophotometry will come into place. Use the blank cuvette …show more content…
The goal in the experiment was to find each protein and the absorbance of each data set. Finalized by creating a graph and finding the slope of the graph. In this experiment the group was able to find the data needed to find the protein and the absorbance. The hypothesis for part two was the amount that the company says is in the milk isn’t the actual amount that the spectrophotometer. The hypothesis for part one was the concentration in each cuvettes was going have little protein and the light would pass
And finally into test tube 3, I pipetted 1.0 ml turnip extract and 4.0 ml of water. The contents of test tube 1 was poured into a spectrometer tube and labeled it “B” for blank. “B” tube was now inserted it into the spectrometer. An adjustment to the control knob was made to zero the absorbance reading on the spectrometer since one cannot continue the experiment if the spectrometer is not zeroed. A combination of two people and a stop watch was now needed to not only record the time of the reaction, but to mix the reagents in a precise and accurate manner. As my partner recorded the time, I quickly poured tube 3 into tube 2. I then poured tube 2 into the experiment spectrometer tube labeled “E” and inserted it into the spectrometer. A partner then recorded the absorbance reading for every 20 seconds for a total of 120 seconds. After the experiment, a brown color in the tube should be observed to indicate the reaction was carried out. Using sterile techniques, any excess liquid left was disposed
1. Fill in the table below with the results from the monosaccharide test experiment, and your conclusions based on those results.
The purpose of this lab was to test different substances using various procedures to see what biomolecules were present and ultimately find out what restaurant Anna Lyza had eaten at before she died. For the first control test, we used vegetable oil to test for lipids. So, if the solution does not contain lipids, it does not become translucent when placed onto a paper bag square and held up to a light. So, it is a negative result. However, in the presence of lipids, the solution will become translucent when placed onto a paper bag square and held up to a light. Therefore in this case, the result is positive. On the other hand, we used albumin egg to test for proteins in another control test. If the solution does not contain proteins, it will not experience any color change and so it is a negative result. When there are proteins existing in the solution, it will turn bluish/purplish and for this reason it is a positive result. Furthermore in the third control test, we used dextrose to test for simple carbohydrates such as glucose. If the solution does not contain simple carbohydrates, it will not undergo any color change and will remain a blue color. So, it is a negative result in this circumstance. If there are simple carbohydrates present in the solution, the solution will turn reddish and so the result is positive. For the last control test, we used starch solution to test
In the experiment we used Turnip, Hydrogen Peroxide, Distilled Water, and Guaiacol as my substances. On the first activity, Effect of Enzyme concentration of Reaction Rate for low enzyme concentration, we tested three concentrations of the turnip extract, and hydrogen peroxide. For the Turnip Extract I used 0.5 ml, 1.0 ml, and 2.0 ml. For hydrogen peroxide we used 0.1 ml, 0.2 ml, and 0.4 ml. We used a control to see the standard, and used a control for each enzyme concentration used. The control contains turnip extract and the color reagent, Guaiacol. We prepared my substrate tubes separately from the enzyme tubes. My substrate tube
The null hypothesis will be that the test tubes with an increase in temperature, pH values, enzyme concentrations, and substrate concentration will have a very small color change or no color change at all. The alternate hypothesis is that the test tubes containing an increase in temperature, pH values, enzyme concentrations, and substrate concentration will all have an intense color change; the more the change, the more intense the color change will be.
The procedures for experiment A, B, and C all start the same. The first step is to put on goggles and get the data collection device set properly. The labquest needs to be plugged into the colorimeter accurately so that a click is heard when putting it in. The labquest needs to be reading digitally and the colorimeter needs to be set to 635 nm. Then shake the chloroplast solution and take a clean cuvette and fill it with 3 mL of distilled water, 3 drops of the chloroplast solution, and cap it. This is used as a blank to calibrate your labquest. Double check that the labquest is reading absorbance, this assures that the colorimeter is plugged into the labquest accurately. Insert the blank into the colorimeter and hit the calibration button. Take out the blank and empty it. The labquest is now set to experiment with. Make sure that the heat bank is set in front of the lamp and that the lamp is on. The cuvette must be placed on the opposite side of the heat bank in the path of light in the box so that no other light can interfere with the experiment.
Colorimetric assay is a process of determining a concentration of a solution based on absorbance of light. The purpose of this lab is to determine if the Bradford assay is an accurate way to determine an unknown concentration of two samples of protein. The Bradford assay is done by measuring wavelength of light passing through a cuvette filled with Bradford dye and concentrations of PBS and proteins. After the cuvettes are mixed they are placed into a spectrophotometer to measure wavelength. The wavelength given will be used to plot a standard curve based on concentration (x-axis) and wavelength (y-axis). The standard curve is then used to measure an educated guess on the concentrations of unknown protein concentrations. We hypothesized that if we use the Bradford assay and colorimetric spectrophotometry we can determine an accurate concentration of two unknown concentrations of proteins. The results of this lab failed to reject our hypothesis based on accurate measurements of protein concentrations. The standard curves are drawn with a linear increasing slope. The Bradford assay is an accurate way to demine the concentration of an unknown concentration.
A spectrophotometer’s purpose is to use colors of the light spectrum to determine the concentration of light absorbing molecules in a solution. (p.59) In this particular lab, our mission was to determine the protein concentration and the standard curve of the unknown sample of BSA. This, by preparing five dilutions of the unknown solution of BSA together with other known concentrations, and then experimenting by observing how the concentrations were passed through the spectrophotometer. The outcome resolved in the absorption levels being decreased, and this
In Exercise 1, diaminofluorene is used to determine the hemoglobin concentration in the daphnids. A higher hemoglobin concentration is indicated by a darker blue color. A spectrophotometer was used to determine the absorbance at 610nm. When measuring the absorbance levels a blank is necessary to have a zero reference, the blank is the “starting point” for the measurements of the sample (re-word). The blank consists of 10µL of diaminofluorene(DAF), 50µL of hydrogen peroxide, and 0.5mL of PBS. The PBS acts as a buffer in this experiment. The cuvette with the sample of daphnids consisted of 10µL of DAF, 50µL of hydrogen peroxide, and 0.5mL of the sample of Daphnia. In Exercise 2, the Pasteur pipette was used to obtain the sample of Daphnia. The depression slide used in this experiment isolated the daphnid, cotton was used to keep the daphnid still while the heartbeat was counted. The ocular micrometer on the microscope allows the tail spine length to be measured accurately, as well as using an ocular magnification table.
2. Four unknown samples were included in the lab kit: flax seed meal, potato starch, egg whites, and dried milk. Using the results of the biochemical testing, determine which number corresponds to the correct unknown. (8 points)
15) Obtain the boiled chloroplast suspension, mix, and transfer 3 drops to cuvette 4. Immediately cover and mix cuvette 4. Insert it into the spectrophotometer's sample holder, read the percentage transmittance, and record it in Table 4.4. Replace cuvette 4 into the incubation test tube rack. Take and record additional readings at 5, 10, and 15 minutes. Mix the cuvette's contents just prior to each readings. Remember to use cuvtte 1 occasionally to check and adjust the spectrophotometer to 100% transmittance.
Apparatus: Spectrophotometer (UV-1201), cuvettes, water bath (set at 37°C), 200µl and 1000µl micropipettes and test tube
* Temperature * Method of measuring solutions I will use equal amounts of trypsin, milk, PH, buffer and distilled water in all the experiments and will use the same measuring equipment to ensure fair representation of the effect of different PH on digestion.
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.
concentration, record the absorbance readings at a fixed wavelength, and plot the absorbance vs. concentration data. The wavelength of 520 nm was selected for experiment Part