Lab Report On Protein Chromatography

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.

PRACTICAL REPORT ON THE ISOLATION AND IDENTIFICATION OF CODEINE AND PARACETAMOLPRACTICAL REPORT ON THE ISOLATION AND IDENTIFICATION OF CODEINE AND PARACETAMOL AIM: To extract codeine and paracetamol from its tablet by solvent extraction and tentatively identify in comparison to standards using Thin Layer Chromatography. INTRODUCTION: Codeine or methyl morphine,

Lab Report 4 Introduction: Protein purification is a process that can be employed to separate a single protein from a larger starting material which may be anything from an organ to a cell. Isolating a purified protein from a larger fraction enables further analysis such as determination of amino acid sequence, potential biological function, and even evolutionary relationship. (Cuatrecasas 1970) In this experiment, the enzyme lactate dehydrogenase will be purified, this enzyme is found extensively in human cells and catalyzes the conversion of lactate to pyruvate, an essential part in energy production. LDH is a key part of anaerobic energy production especially within glycolysis in which LDH catalyzes the conversion of the reverse reaction, pyruvate to lactate, generating NAD+ from NADH, reproducing the oxidized form of the coenzyme which can be used for oxidative respiration. (Markert 1963) Due to the fact that number of purification steps correlates with the purity of the protein multiple purification techniques will be used to isolate a pure form of LDH. LDH will be isolated from a larger “cytosol” fraction collected from a homogenized rat liver in a previous fractionation exercise. Of the procedures that will be used to isolate and purify proteins from a larger fractionate are a set of techniques collectively known as chromatography. These techniques all have the same premise, in that they consist of a stationary phase, also known as the

Enzymes are catalysts that function to speed up reactions; for example, the enzyme sucrose speeds up the hydrolysis of sucrose, which breaks down into glucose and fructose. They speed up reactions but are not consumed by the reaction that is taking place. The most important of the enzyme is the shape as it determines which type of reaction the enzyme speeds up. Enzymes work by passing/lowering and energy barrier and in doing so; they need to bind to substrates via the active. Once they do, the reaction speeds up so much more quickly than it would without the enzyme. Coenzymes and cofactors aid the enzyme when it comes to binding with the substrate. They change the shape of the active site so the substrate can bind properly and perform its function.

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

Roy Levin Bio 11 Lab Dr.Izquierdo Analysis of Macromolecules in Tissue Homogenates of Bos taurusMaterials and Methods The homogenates provided were made by homogenizing tissues in a sucrose phosphate buffer in a 1:20 ratio. The protein concentration in bovine cells was measured by diluting the homogenate with a 1:5 ratio; 50 microliters of homogenate and 200 microliters of water. Then 5 known protein concentration samples which were 0.4, 0.8, 1.2, 1.6, 2.0 mg/ml of bovine serum were used to determine absorbance with a spectrophotometer. Two additional samples were made; one was blank and the other was for the specific homogenate sample. Then 3 microliters of bradford assay reagent, which indicates the amount of protein present

ADAM ANDERSON BIOLOGY SAE01 Experimental investigation into variable heat on Rennin enzyme acitivity and Protien from Milk. 1.1 Experiment on variable Heat effects on Enzyme activity reaction with Protien. Independent variables are the Rennin, Milk solution, and buffer solution. Also time of observations. Dependent variables are the temperature, being 4o, 20-25o, 35-40o, 55-60o and 80-85o. 1.2 I predict

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).

As mentioned in question two, pH is another important factor regarding how active an enzyme could be. Although not all enzymes have the same optical pH levels, a majority of enzymes work the most effectively when the pH of their environment is between six and eight. If most enzymes are put in more acidic or alkaline environment, the enzymes would no longer function and would be irreversibly damaged. For example, the enzymes would not form the proper shape to bond with the substrates or they would not be able to bond with the substrates. As a result, the imbalance of the pH level would inhibit the rate of reaction since reactions would no longer be able to

2. Measure 3ml of 25% catalase concentration to set up for the first trial. 3. Measure 15ml of 10V hydrogen peroxide to set up for the first trial. 4. Using a 5ml measuring cylinder, measure 5ml of the 15ml of hydrogen peroxide, into each of the three measuring cylinders for each trial.

Purpose 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.

Protein standards were previously loaded into the first rows of the well plate of which the concentrations were as followed; 500 μg/mL, 250 μg/mL, 125 μg/mL, 62.5 μg/mL, and 0 μg/mL (PBS only). After making note of the rows used the plates were loaded onto the selected protein row, followed by the addition of 200 μL of Bradford reagent to each well using a multichannel pipette. The absorbance of the plate was read at 595nm and recorded. The NT-2 and LPS-2 protein sample tubes were labeled for easy identification in the following lab and the protein samples were stored in the freezer for 1 week.

Enzymes are catalytic proteins that accelerate the rate of biological reactions while experiencing no permanent chemical modification as a result of their participation in a reaction. In order to initiate a reaction from a reactant called a substrate to a product, a certain amount of energy, otherwise known as the