Purified Protein Lab Report

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.

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

In Figure 3, there is a collected data with the total washes and the elution fraction with the RFUs from the Ni+2 Agarose column. E3 had the highest elution fraction with 8554 RFUs. In the Bradford assay, E3 had the highest protein amount with 65.5ug. The specific activity for E3 was about 1.28E5 RFUs/mg. Overall, in the Figure 3, the E1-E6 had most of the rGFPs due to being bonded in Ni+2. W1-W6 rGFP is much smaller than the E1-E6 amounts.

Introduction: Transformation is used to introduce a gene coding for a foreign protein into bacteria. Hydrophobic Interaction Chromatography (HIC) is used to purify the foreign protein. Protein gel electrophoresis is used to check and analyze the pure protein. Research scientists use Green Fluorescent Protein (GFP) as a master or tag to learn about the biology of individual cells and multicultural organisms. This lab introduces a rapid method to purify recombinant GFP using HIC. Once the protein is purified, it may be analyzed using polysaccharide gel electrophoresis (PAGE).

The objective of this experiment was to verify whether melanoma cells overexpress the p65BTK isoform, as in the case with colon cancer cell lines. Lysates of the melanoma cell lines IGR1, WM266, CHL1, SK-Mel28, MEWO, Colo853, Colo858, SK-Mel3, G361, A375 and C32 (together with a lysate of the colon cell line HCT166p53KO as a positive control) were analysed by western blot as described in the material and methods (3.2.1. Western Blot) using an isoform-specific, homemade antibody raised in the lab. As a loading control the membrane was also probed with a commercial antibody against the housekeeping gene GAPDH, encoding an enzyme important for the glycolysis.

This secondary antibody was used for both halves of the membrane, because both primary antibodies were made from rabbit. In theory, a LDH band should be observed in each of the sample 1~4, because all four samples contain LDH enzyme. A band was observed for each well 1,2 and 4, which suggests that LDH enzymes are present. Sample 4’s band however, is very faint.

For the second part of the experiment, one had to use the knowledge learn from viewing protein molecules in FirstGlance in Jmol to analyze the protein PDB ID: 4EEY. The analysis of this protein was done using the RSCB protein data bank (PDB) at (http://www.rcsb.org/pdb/home/home.do).2

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

Many work has been done to perform an integrated analysis using genetic and gene expression data.

Immunoblotting is an effective approach that utilizes antibody-tags to analyze, isolate, and quantify proteins. A Western Blot, a type of immunoblotting method, was used in this experiment to evaluate the protein quantities contained within the different subcellular fractions. Although, the Western blotting method can be implemented for the study of more complex cellular processes. Complex cellular processes such as metabolism can be greatly evaluated with immunoblotting methods. For example, some of the players involved in enzymatic activities in cellular metabolism can be identified with immunoblotting techniques. (Lewin, Van Horn, Krisans, & Coleman, 2002) To analyze the Western blots obtained in this experiment, the varying fraction bands

There were several experimental procedures that were performed by Bayry et al that allowed for this experiment to be performed. One of these procedures included treatment of an IgE antibody that is produced specifically in mice, SPE7 with heme using the technique immunoblotting to determine the polyreactivity of heme and the resulting consequences of the reaction. Immunoblotting, also known as western blotting, is a type of an assay that is specifically used for the detection and characterization of proteins1. According to Gallagher and Chakavarti, immunoblotting works by exploiting the specificity inherent in antigen-antibody recognition5. It involves the solubilization and electrophoretic separation of proteins, glycoproteins, or lipopolysaccharides by gel electrophoresis, followed by quantitative transfer and irreversible binding to nitrocellulose, PVDF, or nylon5. The immunoblotting technique has been useful in identifying specific antigens recognized by polyclonal or monoclonal antibodies and is highly sensitive5. This unit provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization5. Another technique used was an Enzyme-Linked Immunosorbent Assay or ELISA. The purpose of an ELISA is to determine if a particular protein is present in a sample and if so, how much6. There are two main variations on this method and whichever one is used depends on the type of experiment that is being run. One can determine how much

The induction process for the activation of rGFP was vital to this experiment, to ultimately determine the presence and molecular weight of rGFP from crude extract. T7 RNA Polymerase activates the T7 Promoter, by binding to it upstream of the rGFP gene, in the presence of IPTG, allowing the expression of rGFP. The expression results from the transcription of the rGFP gene that produces rGFP mRNA, which is then translated to produce the rGFP. By inducing the IPTG activation of the DNA regulatory sequence T7 Promoter, as seen in Figure 1, the presence of rGFP became apparent under the UV lights after the Ni+2 Agarose Affinity Chromatography washes and elutions. The G3 sample, three hours post-induction of the IPTG sample, had the highest fluorescence at 14901 RFUs.

Cells were lysed with Laemmli buffer, and each lysate sample was loaded into a polyacrylamide gel and separated by electrophoresis at 30 mA following by transfer onto PVDF membranes (Millipore). Membranes were blocked for 1 h at room temperature using 5 % skim milk in Tris-Buffered Saline with 0.1 % Tween-20 (TBST). WB analysis was

In order to produce crude extract, bovine tissue was obtained, precisely minced to exclude extra fat, and then blended with pH 7.2 phosphate buffer. The purpose of blending the tissue with the buffer was to pulverize the cells, causing them to release their contents evenly—most importantly lactate dehydrogenase (LDH)—into the solution. Since other cell components such as proteases which reduce LDH were also released from the lysed cells, the slurry was kept on ice to minimize their kinetic activity. The solution was then centrifuged, creating a pellet made of cellular debris and leaving behind the crude product in the supernatant. Using data from the enzyme assay, the crude extract revealed 155±7 mg total protein, 4980±80 units of total activity, 32±2 units/mg of specific activity, a 100% yield, and a purification factor of 1 fold (Table 1).

En general la base de datos provee información sobre las funciones que realiza cada una de las proteínas "moonlight" disponibles en la base de datos junto con su NCBI y UniProt "accesion number", el estado oligomerico y monomerico de la proteína, código PDB y referencias bibliográficas sobre las funciones designadas a cada proteína. Dentro de las características de la base de datos hay que mencionar que los desarrolladores nombraron como función canónica a la primera función que fue descubierta de la proteína y "moonlight" a la segunda función que haya sido descubierta de la misma proteína. Multitasking Proteins DataBase posee un formato y diseño sencillo que facilita la obtención de información sobre las proteínas moonlight disponibles en la base de datos.