Rubisco Lab Report

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

Protein Assay: The Pierce BCA Protein Assay (Thermo Scientific) is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein concentration. A series of standard solution of Bovine Serum Albumin (BSA) ranging from 0-2000 µg/ml was prepared from a stock solution of 2 mg/ml BSA. 25ul of diluted crude (1:500, 1:250), desalted (1:100, 1:50), and 6 peak fractions from cibarcon blue column (1:10, 1:5) were loaded in microplate along with 175ul of BCA working reagent. Microplate was incubated for 30min at 370C and then the absorbance was measured at 562nm.

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.

Rubisco is the most abundant protein on earth that is essential for carbon fixation in plants. For the protein to function at its optimal level, it needs to be isolated from the mixture of proteins and in its purest form. The three isolation techniques carried out in this lab are salting out, ion exchange chromatography, and SDS-PAGE. Rubisco will be purer as each technique is conducted and will be in its purest form after the last isolation technique is carried out.

In this project, C. Elegans are hermaphrodite worms that will be used since they are easy to maintain in lab, as well as have short life cycles. The gene that the project attempted to knockdown in C. Elegans with RNAi treatment is the unc-22 gene. RNAi disrupts gene expression in the presence of double stranded RNA (dsRNA) that is complementary to target gene sequence. The unc-22 gene codes for a muscle protein called twitchin in wild-type worms. The Unc-22 is required for muscle regulation and maintenance in C.Elegans. To verify that the RNAi treatment worked, would check the unc-22 mRNA levels in the worms, in addition to phenotype observation.

The proteins are also added to a Laemmli sample buffer in order to give each protein a negative charge so it is able to get pulled through the polyacrylamide gel. The next step is to put the gel into the electrophoresis module and to run it. It is run until the proteins have almost reached the bottom of the gel. A blue tracking dye is added to the Laemmli sample buffer in order to track the distance in which the proteins travel through the gel. If it is run for too long, the proteins will run off the bottom of the gel and it will mess up your results. Once the protein reach the bottom of the gel, the gel is stained in order to be able to see the individual bands of the different proteins. When the gel is stained, the protein distances will be able to be measured and compared. For a detailed procedure, refer to the Comparative Proteomics Kit I: Protein Profiler Module Lab Manual.

Western blotting - In Western blotting first, the macromolecules have to be separated via gel electrophoresis. The molecules now separated by electrophoresis are blotted onto either a nitrocellulose or a polyvinylidene difluoride (PVDF) membrane (a second matrix). To inhibit the binding of nonspecific antibodies to the membrane surface it is subsequently blocked. Then a complex is formed (a probe) from the protein that was transferred and an enzyme linked with an antibody. The enzyme is supplied a substrate then the 2 together should create a product e.g. chromogenic precipitate that can be detected. Detection methods with most sensitivity use chemiluminescent substrate because light is a by-product of the reaction between the substrate and the enzyme. The output of the light can be measured using a CCD camera or on the other hand, antibodies that have been tagged with fluorescents that are detected with a fluorescence imaging system can be used (Thermo-Fisher Scientific 2015).

The enzyme behind this life-changing innovation is Ribulose-1,5-bisphosphate carboxylase/oxygenase, better known as Rubisco. Rubisco is involved in the first major step of carbon fixation, a process by which atmospheric CO2 is converted by plants and other photosynthetic organisms to energy-rich molecules. In chemical terms, rubisco catalyzes the carboxylation of ribulose-1,5-bisphosphate (also known as RuBP) (“Ribulose 1,5-Bisphosphate”). This begins three rounds of a cyclical series of reactions in which three CO2 molecules are converted into a compound called glyceraldehyde-3-phosphate, which is converted into various nutrients. RuBP is replenished at the end of each cycle. The used up ATP and NADPH are converted to ADP and NADP+, respectively, which are used for the light-dependent reactions (Cranford).

Biochemical Journal , 4 (3-4),

In the Affinity Chromatography experiment we were purifying our Con A proteins. In general, affinity chromatography is a technique that is used for isolating a protein, in our case Con A from a large amount of other macromolecules. Our protein of interest is captured using a microbead matrix while we let everything else flow through the column. The Sephadex matrix is made of cross-linked glucose or dextran and because our Con A has an affinity for glucose it is able to bind to those beads. In general, we began by equilibrating our column with NaCl, then poured Jack Bean Meal Extract which so happens to contain Con A through our column, the Con A then binded to the Sephadex beads, and finally we eluded with a dextrose solution so that

P1 and P2 centrifuged for three minutes at 1000rpm. Supernatant transferred to Eppendorf tubes, 1ml of each saved and set aside. P1 diluted by a factor of 100 and loaded in a column with 5mL. 5mL undiluted P2 loaded into a separate column. 10mL Buffer A used to wash the column. A 10mL of low-salt buffer loaded into each column, 1-2mL collected into each cuvette. Cuvettes scanned with a spectrophotometer, blanked with low salt buffer. Fraction contained the most protein identified and isolated into an Eppendorf tube and placed on ice. The same procedure followed for medium salt and high salt, the blank correlated with loaded buffer. The beads cleaned with a 10mL resin cleaning buffer.

The enzyme solution was strained through a layer of cheesecloth and the solution was funneled into a container. The use of cheesecloth allowed for liquid to pass through while separating larger particles and materials. The container was sealed to limit the amount of oxygen entering the solution and kept on ice to preserve the enzymes of the solution. v. A spectrophotometer was obtained and turned on.

The purpose of this experiment was to isolate two different proteins, from an availability of three (Myoglobin, BSA or Cytochrome C) using an ion-exchange chromatography method via a cation (CM) or anion (DEAE) exchanger. This was followed by a SDS-PAGE gel electrophoresis technique to determine the original mixture’s proteins and concentration with the help of a Bradford assay, which utilizes a Coomassie dye to bind to proteins. After all techniques were performed, Protein 1 was found to have an absorbance value of 0.451 and a concentration of 0.019 µg/µL. Protein 2 was found to have an absorbance of 0.373 and a concentration of 0.016 µg/µL. Ultimately, using the anion exchanger (DEAE), negatively charged proteins attached onto the immobilized

A glass column with inner diameter of 1 cm, was packed with Bio-Gel P-100 (BIO-RAD) beads suspended in equilibrium buffer of 20 mM phosphate buffer with a pH of 7.0 - 7.4. The height of the total content was kept at 5cm. The sample mix with total volume of .1ml contained 2 mg/ml Blue Dextran (blue, 2 MDa), 5 µl yellow food coloring (yellow, ~500 Da), 2 mg/ml Hemoglobin (red), 2 mg/ml of BSA in phosphate buffer. As soon as the sample mix was added to the column, 0.25 ml (~2 drops) of fractions were collected in the Eppendorf tubes until all the colored bands eluted the column because it was assumed that the colored band represented protein content. Approximately 1 µl of each of the fractions collected were spotted on to nitrocellulose filter using capillary pipet. After the filter was dried, it was incubated in 50% methanol for 5 minutes then it was incubated in Amido Black (0.1% amido black 10B in 30% methanol) for 5 minutes. Next, the filter was

Affinity chromatography relies on the protein ability to bind to specific molecules tightly but not covalently. This technique uses a ligand bound to the matrix that is capable of specifically binding to the protein. When the impure solution is passed through the