Western Blot Analysis

This Lab Report is an analysis of the results of a two-part experiment. In the first part, we used a gel filtration column to separate the components of a mixture composed of protein and non-protein molecules. By doing so we hoped to obtain fractions that contained single components of the mixture, while also gaining insight into the relative molecular weight of each component compared to each other. We would then plot these fractions onto nitrocellulose paper in order to determine which fractions had protein. In the second part, we would use the fractions which we had determined had protein to conduct an SDS-PAGE. By doing so we hoped to determine an estimate on the molecular weight of the proteins present in each fraction by comparing it to a tracker dye composed of a variety of molecules of differing molecular weight.

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.

Gelatin is observed to have a lower absorbance reading than lysozyme in only the Bradford assay, while in the BCA assay gelatin was observed to have about the same absorbance as lysozyme. In Bradford assay, the color yield for proteins with higher content of tryptophan, tyrosine, or cysteine residue, will be higher. The observed protein sensitivities did not correlates with the amino acid content in the Bradford assay. As presented in Table I, the mole fractions of lysine and arginine in BSA were 10 mole percent and 4 mole percent, respectively. The sensitivity should have been higher in BSA than in the Bradford assy. However, the observed sensitivities did correlates with the amino acid contents in the BCA assay.

1. EMG 9 and EMG 26 contain strain _lac-_(I- Z+ Y+) and strain _lac -_ (I+ Z- Y-)respectively.Three genes huddled together on the chromosome are required for two strains of _E.coli_ to utilize lactose.Consisting of three genes, namely, _lacZ_, _lacY_ and _lacA_, the _lac_ operon orderly handles these genes to code specific enzymes necessary for the metabolism of lactose. The genes _lacZ_, _lacY_ and _lacI_ would code for beta-galactosidase, galactosidase permease and _lac_ repressor respectively. Regulation of _lac_ operon is also tight and the operon's negative control is made possible by _lac_ repressor (Hill, 1996). With the presence of _lac_ repressor, _lac_ operon will be deactivated and will

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

ELISA works on the principle of an antigen binding to specific antibody (lock and key), which can be used as a way to identify quantities of proteins in a small sample of fluid. The specific proteins used in an ELISA are estimated quantitatively. The ELISA test is carried out by incubating the serum that contains the antigen of interest with antibody’s within a well, in order for the antibody’s to bind with the specific antigens. The plate is then washed with a mild detergent in order to remove any proteins that have not been bound. The washing of the plates is carried out between every step in order

A: The Bovine Albumin Serum ‘BSA’ is a protein derived from cows and is commonly utilised in ELISA tests to prevent non-specific binding from occurring. Conversely, it also serves as a protein supplement. The BSA is

Western blotting is used to tell you how much protein has accumulated. It takes a protein that has been separated via gel electrophoresis and transfers it onto a blot which is then labeled with antibody and immunostained in order for the antigen bands to be visualized.

In cross B, a red-eyed female fly was placed into a vial with a white-eyed male fly and a red- eyed male fly for a breeding period of two weeks. At least eight red-eyed females were produced from this cross. These eight females were used to conduct a polymerase chain reaction. Comparing to the control groups, the results show that seven out of eight flies has the Xw+ Xw genotype because all of them had both bands appear on the gel when both alleles were present. This indicates that all of them are heterozygous. They are all red-eye females. Fly 7, however either of the band appear. This means that an error occurred, and one possible error could be not enough DNA were extracted therefore the gel could

The main purpose of this lab is to identify and separate the main components of milk. To do this an understanding of the properties of these components in needed to separate them from one another. We will be separating the components with their polarity or non-polarity and the temperature at which specific components precipitate. To do this we will be using hot plates, gravity filters and vacuum filters1, water and ice baths, and blot drying.

All inoculated ewes at autopsy had BTV antibodies. The BTV-1 control ewe also developed antibodies. BTV-8 infection induced mild clinical signs where BTV-1 infection developed more severe signs. BTV-8 was demonstrated in 43% of fetuses where BTV-1 was demonstrated in 82%. Pathological changes were found in the central nervous system and in the

The polyacrylamide gel showed bands of proteins based upon their weight. The further away from the gel the protein band occurred, the heavier the protein was. The Precision Plus Protein Kaleidoscope prestained standard and actin and myosin standard ladders were very neatly spaced out, and one could easily detect the presence of different proteins. However, the fish proteins were not so easily discerned, especially since, in most cases, the lighter proteins had less color.

1Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical sciences, Texas A&M University, College Station TX. 2 Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Valencia, Burjassot, Valencia, Spain.

Western Blotting can be used to detect the Myosin actin light chain in different species of fish and is used to distinguish from different species based on variation, commonality, or evolutionary divergence. First, proteins are extracted from the tissue and loaded into a gel matrix. The matrix will separate the proteins according to size using an electric current. Proteins that are separated after are blotted from the gel and onto a paper membrane. An antibody is then added to the membrane paper and causes a colored reaction. Following the reaction, the results

Milk proteins such as whey and casein are isolated for many different functions in food systems such as: foaming, whipping, gelation,