Report on Result and Discussion on isolation of protein from spinach leaves
In this experiment, we tried isolating an actual protein called Rubisco from spinach leaves. The full meaning of these Rubisco means Ribulose-1, 5-bisphosphate carboxylase/oxygenase which is an enzyme that catalyzes the addition of a carbondioxide group to ribulose-1, 5-bisphosphate during sugar production in green plants. Although Rubisco is an abundant protein in plants, it is not the major component in all spinach cell and to successfully extract these protein putting into consideration the solubility, the charge and size we had to use different purification techniques to separate this
Single protein from the intricate mixture of proteins and other materials that …show more content…
One SDS-PAGE Gels were used to separate the proteins. The gel was transferred onto a membrane via electrophoresis. We did not know how much protein that was in each sample; however we can assume that the protein concentrations were low; hence 25-30 µL of each protein samples was added to each lane. Figure 2. The standard ladder for analyzing the SDS- page and SDS-PAGE acrylamide gel after staining with buffer containing bromophenol Blue, glycerol, dithiothreitol, and SDS.
Lanes 1 2 3 4 5 6 7 8 9 10
Filtrate S1 P1 P1 medium salt P1 high salt S2 P2 P2 medium salt P2 high salt Loading Dye
Table 3: showing what was inserted in each lane in the gel.
Table 4. A) Showing the data collected in sample 2 after measuring size (kDa) of the band shown in gel, getting the distance (mm). B) The log of Size used to construct a standard curve.
Size (kDa) Distance
97 10
66 18
45 22
30 25
20.1 30
14.4 35
A) Up and B) down
Log of …show more content…
Ammonium sulfate precipitation, ion exchange chromatography, and SDS-Page were the three different techniques used in this experiment. The standard molecular weight of rubisco is 66 kDa and 14 kDa. rubisco was expected in P2 with nothing added. Also in P2 medium salt the evident of rubisco. T The standard coefficient of rubisco is between 14.1 ug/mL and 18.2 ug/mL (Hudson, 1994). The average of the two numbers, which is 16.15 ug/mL, was used to calculate the protein concentration in each sample in tables 4-7. Pellet two with nothing added did not have a protein concentration because the OD value at 280 nm was a negative number, which is, calculated the same as zero. Even though we got a good image of the SDS-PAGE under the Ultraviolet light still does not mean that data collected are accurate; however, three bands wear found on the SDS-page which was read and the result. Most error were as a result of not adding enough buffer or not placing the solvent sin each lane properly which can hinder us from getting accurate data so as to make sound decision. According to our results, it is evident that Rubisco was being success fully isolated using different technique. These results obtained are important because they tell how much rubisco is anticipated in the different sequence of solutions prepared and used in effectively performing the experiment. Also getting a good SDS-Page didn’t mean we did
In this experiment, we tried isolating an actual protein called Rubisco from spinach leaves. The full meaning of these Rubisco means Ribulose-1, 5-bisphosphate carboxylase/oxygenase which is an enzyme that catalyzes the addition of a carbondioxide group to ribulose-1, 5-bisphosphate during sugar production in green plants. Although Rubisco is an abundant protein in plants, it is not the major component in all spinach cell and to successfully extract these protein putting into consideration the solubility, the charge and size we had to use different purification techniques to separate this
Single protein from the intricate mixture of proteins and other materials that …show more content…
One SDS-PAGE Gels were used to separate the proteins. The gel was transferred onto a membrane via electrophoresis. We did not know how much protein that was in each sample; however we can assume that the protein concentrations were low; hence 25-30 µL of each protein samples was added to each lane. Figure 2. The standard ladder for analyzing the SDS- page and SDS-PAGE acrylamide gel after staining with buffer containing bromophenol Blue, glycerol, dithiothreitol, and SDS.
Lanes 1 2 3 4 5 6 7 8 9 10
Filtrate S1 P1 P1 medium salt P1 high salt S2 P2 P2 medium salt P2 high salt Loading Dye
Table 3: showing what was inserted in each lane in the gel.
Table 4. A) Showing the data collected in sample 2 after measuring size (kDa) of the band shown in gel, getting the distance (mm). B) The log of Size used to construct a standard curve.
Size (kDa) Distance
97 10
66 18
45 22
30 25
20.1 30
14.4 35
A) Up and B) down
Log of …show more content…
Ammonium sulfate precipitation, ion exchange chromatography, and SDS-Page were the three different techniques used in this experiment. The standard molecular weight of rubisco is 66 kDa and 14 kDa. rubisco was expected in P2 with nothing added. Also in P2 medium salt the evident of rubisco. T The standard coefficient of rubisco is between 14.1 ug/mL and 18.2 ug/mL (Hudson, 1994). The average of the two numbers, which is 16.15 ug/mL, was used to calculate the protein concentration in each sample in tables 4-7. Pellet two with nothing added did not have a protein concentration because the OD value at 280 nm was a negative number, which is, calculated the same as zero. Even though we got a good image of the SDS-PAGE under the Ultraviolet light still does not mean that data collected are accurate; however, three bands wear found on the SDS-page which was read and the result. Most error were as a result of not adding enough buffer or not placing the solvent sin each lane properly which can hinder us from getting accurate data so as to make sound decision. According to our results, it is evident that Rubisco was being success fully isolated using different technique. These results obtained are important because they tell how much rubisco is anticipated in the different sequence of solutions prepared and used in effectively performing the experiment. Also getting a good SDS-Page didn’t mean we did