Macromolecular Analysis

Perform a series of accurate tests on biological molecules to detect the presence of carbohydrates and proteins, as well as the action of an enzyme on specific molecules.

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

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.

We placed the gel into the running chamber, and then we completely covered the gel with TAE. 3 microliters of loading dye was added to each tube; this would help distinguish the enzyme from the gel. As before, we tapped the tube on the table to mix. Then we carefully added each of the four samples into their own wells. A total of 33 microliters of each sample was poured into each well. Afterwards, we attached the positive and negative electrodes to their corresponding terminals on the power supply and gel box. We turned on the power to around 80 volts and waited 45-60 minutes for the loading dye to move down the gel approximately 6-8 cm. Finally, we were able to visualize the DNA in the gel and write down the

1. Carbohydrates: Are Molecule composed of carbon, hydrogen, and oxygen, and they include sugars and starches.

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).

Proteins are important elements in cellular membranes and give the membranes many of their characteristics. In red blood cells, the meshwork of proteins in and around the membrane gives it strength and flexibility, allowing a cell to squeeze through small capillaries without bursting. Other proteins play roles in transporting material in and out of the cell (Lab Manual, Cell Biology). Polyacrylamide gel electrophoresis (PAGE), with all of its different modifications is probably the most widely-utilized procedure in contemporary biochemistry and molecular biology (Mordacq and Ellington (1994)). In this experiment, we will attempt to determine the molecular weights of the major proteins in the plasma membranes of bovine red blood cells (RBCs). The predictions made are if our protein has similar weights as proteins

Gel electrophoresis is a procedure used in laboratories to separate DNA, as well as RNA and proteins. A gel slab is placed in a buffer-filled box and an electrical field is applied. The negatively charged DNA will migrate towards the positively charged side, where it can then be recorded and further analyzed.

Lab Report Title: Testing Spam for the Presence of Certain Macromolecules Purpose: In “Mystery Meat Revealed”, the purpose was to find the hidden macromolecules ingredients in SPAM, such as starches, simple sugars, proteins, and lipids. A.Curricular Purpose: In class, we are currently studying unit 1, biochemistry, which includes macromolecules, bonds, and characteristics of water. This lab relates to our current unit because we are trying to find what macromolecules are present in SPAM; such as: lipids, sugars, starches, and protein.

In the laboratory, we are testing the DNA of our two suspects, and the DNA sample from the crime scene. My lab assistants and I will be using an agarose gel to analyze the DNA. We will first dissolve the agarose powder by heating it in a buffer, and then pouring it into a casting tray to harden. Small pores will form as the gel hardens, and they will act to separate the DNA samples. Next, the cooled gel is placed into an electrophoresis chamber. It will be covered by a 1xT TAE buffer. We will load the DNA samples into the compartments and the electrophoresis chamber will be plugged into a power supply. When the power is connected to the chamber, electric currents will move through the agarose gel. The DNA fragments are negatively charged, so when placed into an electric field, they will be attracted to the

Agarose is a polysaccharide that is used especially in gels for a process called electrophoresis. It is used in DNA fingerprinting because it slows down the speed at which the DNA migrates from the negative pole to the positive pole so that the process is easier to visualize and to gather results from. In addition to this, the agarose also creates the actual gel that is being manipulated.

This experiment overall was very successful as using information obtained from the results of both the single and double digests, a credible restriction map for the unknown plasmid could be constructed. Within this experiment, both single digest and double digests consisting of three restriction endonucleases were used in order to map out the restriction sites of the enzymes making up an unknown plasmid. In order to separate the DNA fragments by their distinct number of base pairs, it was necessary to run an agarose gel electrophoresis. For this particular experiment, a 1% agarose gel was used as this concentration ultimately results in pores that can separate the DNA by size. The process of gel electrophoresis is made possible by the electric current that is used to move the samples of DNA throughout the gel. For this particular experiment, the gel was run at a current of 100 volts. As a result of the phosphate backbone of DNA, DNA is negatively charged. Because DNA is negatively charged, it moves away from the negative electrode and moves down the gel toward the positive electrode as it is attracted that way. What allows for the resistance the fragments face when moving away from the negative electrode is the texture of the gel itself. As a result, the slower fragments of DNA have the ability to move at a faster rate than the larger fragments of DNA. Each sample loaded into the gel contains a loading-dye for two reasons. One reason is that the loading dye contains

Agarose Gel Electrophoresis refers to the movement of a charged particle in an electrical field, usually performed for analytical purposes, and is routinely used for the preparation and analysis of proteins, and DNA – often after amplification of DNA. It is a procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field. DNA is negatively charged and when placed in an electrical field, DNA will migrate towards the positive pole (anode). An agarose gel is used to slow the movement of DNA and thus separates the size of molecules based on their rate of movement through the gel under the influence of an electrical field.

For this experiment we also made use of agarose gel electrophoresis, which takes a lot of time. Electrophoresis may be the main technique for molecular separation in today's cell biology laboratory. In spite of the many physical arrangments for the apparatus, and regardless of the medium through which molecules are allowed to migrate, all electrophoretic separations depend upon the charge distribution of the molecules being separated.