Analysis Of Two Dimensional Electrophoresis ( Ief )

After you have reviewed the principles of electrophoresis, use what you know to complete the following:

Major scientific concepts in this lab include chromatography and pigments. Chromatography is the division of mixtures, solution, and others into its different components. Chromatography is often used to separate a solution into its separate parts. Chromatography filters complicated compounds accurately without involving a difficult process so it is often used. In this lab, we used chromatography to separate the different pigments in the plants. When looking at plants, the colors of the plants are the colors that are reflected from the different wavelengths of the light spectrum. The colors that are not evident are the colors the plants absorb and use as energy. Pigments are the compounds of a plant the enable the reflected colors to be evident. These pigments function to capture light energy from the sun to enable the plants to undergo photosynthesis.

This technique separates Rubisco samples based on their size. The electrophoresis has a positive and a negative end. Positive charge proteins are loaded from the positive end and migrate towards the negative end. Negative charge proteins are loaded from the negative end and migrate towards the positive end (Sakthivel & Palani, 2016). The sample that contained the highest molecular weight of Rubisco will travel the shortest distance on the gel while the protein with the smallest molecular weight will travel the longest distance (Sakthivel & Palani, 2016). The size proportion of each Rubisco molecule correlates with the distance traveled. Rubisco will be in its purest form after running through SDS-page since each technique will increase the purity of the protein. If the salting out, the ion exchange and the SDS-page protein isolation techniques are performed on protein Rubisco, then it is purified and separated by solubility, charge, and size. The rationale of this experiment is to isolate the purest form of Rubisco so that it can perform carbon fixation at an optimal

The purpose of this experiment is to practice common organic laboratory techniques inside the lab to get one oriented to the basic methods of procedure that can be used for later experiments. This experiment involves the separation of benzoic acid from a more crude form, consisting of benzoic acid, methyl orange, a common acid/base indicator, and cellulose, a natural polymer of glucose (Huston, and Liu 17-24). The technique that is used to perform this separation is called extraction. Extraction is a systematic process of separating mixtures of compounds, taking advantage of the affinity differences of compounds to separate them (Padias 128-37). This technique recognizes the principle that “like dissolves in like,” that is,

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.

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.

· Explain the process of electrophoresis as it relates to separating and identifying different DNA fragments

Extraction technique also cannot be used to separate these two compounds because of the same property they have: they are nonpolar, resulting non acid-base extraction. Extraction technique is used to separate two different compounds to organic and aqueous layer, which means one phase of one solvent has to be polar and the other phase has to be nonpolar.

Buffer is poured into the electrophoresis box, usually a horizontal acrylic container. The gel, still in the mold, is placed into the buffer inside the box. The gel is slightly submerged in the buffer which will conduct the electrical current in the gel. With a micropipette and a new pipet tip, a loading buffer, which increases the density of the sample, is added to the DNA sample in a tube. The DNA sample is colorless, so a tracking dye, such as bromophenol blue or xylene cyanol is used to visually track the DNA movement. The dye migrates at a specific speed similar to the DNA. The sample is then transferred into the first well in the gel. With a new clean

60uL of each sample transferred to Eppendorf tubes, added with a 30uL sample buffer and heated at 95-degree Celsius for 4 minutes. The electrophoresis apparatus set up and filled with Tri-HEPES SDS running buffer. A 25uL of each sample loaded in this order from right to left: ladder, filtrate, S1, P1, P1 medium salt, P1 high salt, S2, P2, P2 medium salt, P2 high salt. The gel ran for 50 minutes at 110V and analyzed with UVP analyzer.

You can view macromolecules by using a technique called gel electrophoresis. Gel electrophoresis is a process used by scientists to study and view macromolecules. This process separates the different macromolecules and coats the different macromolecules so you can easily see them. A macromolecule is a very large molecule that contains many atoms. Agarose is used in gel electrophoresis to coat the macromolecules.

Liquid-liquid was the isolation technique that was utilized in this experiment. The acidified reaction mixture was added to a separatory funnel along with

Lab 8: Investigation 5 Lab question: How do you separate molecules that are attracted to one another? Procedure: Place a small amount of solvent in separate beakers. Then place the mixture on chromatography paper and put the paper in each beaker. Controls:

The cells are placed into a flask and are forced through a nozzle so small that they must pass through one by one. In the nozzle, the cells are vibrated at different frequencies to produce drops (3). The drops of cells are then scanned by a laser that is used to count and measure each cell. Separating populations of cells involves attaching antibody linked fluorescent dye to certain cells of interest (3). The information that is gathered from the sorting and measuring of the cells is evaluated by a computer. The final steps for the FACS include applying an electrical charge to the drops of cells (3). Before the drop of cell forms at the end of the nozzle, a charge is applied to the stream that will determine where the drop will go (3). Based on the charge, the drip is either moved left or right with electrodes or placed in to designated final tubes. Quantifying the FACS information involves displaying the information so we know how many cells of each color and charge were

Chromatography is a separation technique in which the mixture to be separated is dissolved in a solvent and the resulting solution, often called the mobile phase, is then passed through or over another material, the stationary phase. The separation of the original mixture depends on how strongly each component is attracted to the stationary phase. Substances that are attracted strongly to the stationary phase will be retarded and not move alone with the mobile phase. Weakly attracted substances will move more rapidly with the mobile phase.