Bovine Serum Albumin

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

The specificity of albumin binding experiment was to determine the binding interactions that occur between serum albumin and three synthetic dyes with the use of electrophoretic procedure. Whole blood, or plasma. Clots upon standing and if the clot is removed, the remaining straw colored fluid is called serum. The major protein in serum is albumin which functions as a carrier molecule for the transport of certain small molecular weight compounds in blood. Molecules that bind to serum albumin are fatty acids, hormones and some synthetic dyes. In this experiment the synthetic dyes used are Bromophenol Blue, Ponceau S and Orange G. we observed that free dyes not bound to albumin migrate faster that albumin or dyes bound to albumin. This

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.

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).

A clarified and physiologic buffered (pH 7 to 8) is added to the antibodies and purified ZA2GP to form a mixture that is added to the immobilized ligand.

This was done by stacking a sponge, and filter paper behind a gel with nitrocellulose, with the two gels facing each other, then was all compressed between a locking cassette lids. Once transferred the bands were stained with for 2 minutes with 20 ml of Ponceau S then rinsed with distilled water until the bands appear just right. Next, the membranes were placed back to back with protein facing outward from eachother and was incubated for 30 minutes in 30 ml of 5% non fat dry milk/TBS. after the 30 ml was discarded, 7ml of the Primary antibody, mouse IgG anti Xpress epitope MAb solution was added and incubated with the shaking platform for 45 minutes. Then in triplicate the membrane was washed with 30 ml of 0.05% tween20/TBS and shaking for 5 minutes. 7ml of a secondary antibody, (sheep IgG anti-mouse IgG conjugated with horse radish peroxidase polycolonal antiserum), (antibody diluted 1:1,500) was added and incubated on a shaking platform for 45 minutes. Then perform the wash steps again in triplicate, but this time do a fourth final wash with TBS and incubate on shaking platform for 5 minutes like the previous washes. Finally The membrane was developed by the addition of TMB, and rocking the solution back and forth until the bands were apparent, then the membrane was put into water to stop he reaction, token out dried and the results were

For the peptide-based drugs it is a rate-limiting step to cross the BBB. Chimaric peptides contain the drug in brain but are unable to cross the BBB so; we conjugate the drug with targeting vector. Now, these Chimaric proteins can easily pass through the BBB and can be detected by fluorescent markers in the

Second, for the western blotting, the primary antibodies that used to recognize the protein of interest (FLAG-PTEN) are rabbit anti-PTEN antibodies and the secondary antibodies that bind to the primary antibodies and convalently linked substrate to produce visible signal are goat anti-rabbit conjugated HRP (horseradish peroxidase) antibodies; for immunoprecipitation, the antibodies used to immobilize protein of interest are anti-FLAG monoclonal antibodies from morine cell. As introduction above, those antibodies come from different species. In order to be successful in western blotting, the primary antibodies recognize the protein of interest as well as bind to it. Consequently, rabbit anti-PTEN antibodies were used to bind to FLAG-PTEN. Meanwhile, goat anti-rabbit conjugated HRP antibodies, the secondary antibodies,

This is because inhibition of fibril-dependant secondary nucleation can prevent neurotoxin production and possibly be used as immunotherapy for neurodegenerative disease treatment. To carry out the experiment amyloid-B 42 residue was purified, as the monomer and fibril were isolated. Single-chain antibody fragments (scFvs) were selected with a high affinity for AB42 fibrils. Affinities were measured with surface plasmon resonance. They finally used kinetic screening and analyzed using microscopy. From this, they identified four single-chain antibody fragments that specifically inhibit the fibril-dependant secondary nucleation. They also suggested from this that Hydrogen bonding is important in fibril binding specificity. These experiments were In vitro only, therefore in vivo work should be done to see any off-target effects or determine therapeutic efficiency. This work can be further applied for immunotherapy treatment development for various neurodegenerative

Candidate Provines was very effective in receiving the order from higher and translating that into his own order, utilizing the five paragraph format, to his fire team. Of note, SNC was able to take the mission statement from higher, which was void of a ‘IOT’ statement, and develop his mission statement to his fire team utilizing the proper 5 W’s and IOT format, which showcased his grasp of the orders format and the effectiveness it provides the commander. The rest of his brief followed the proper orders’ formatting but failed to include a plan during SOM. It was just a reiteration of coordinating instructions. However, SNC’s tasks displayed that Candidate Provines had developed an initial plan to tackle this problem. Once in execution, SNC showcased drive and determination to get his team across the bridge. Even when members had to be assessed penalties for touching red areas, SNC was able to recognize the time delay and utilize the team member in security to replace that penalized member. Candidate Provines proved to be effective in this means of controlling and utilizing his team members to address the friction of this problem. With a shortage of time, SNC’s team failed to reach the

. To measure the CH50 (i.e. the concentration of serum at which 50% of cells are lysed) of sheep erythrocytes as a result of the introduction of carp serum.

What are cows exactly? Some people would say they are meat or milk producing animals. They would be almost correct. The cow otherwise commonly known as the bovine has many different classifications. The bovine are classified as a type of large domestic ungulates. They are members of the sub family Bovinae and are in the Binomial name of Bos Taurus. (1) The correct term when speaking about cows is to use the term cattle. A cow is a singular word for one of many. Cattle is also not the original term for the bovine. It was a term that had been borrowed from a man named Anglo-Norman. Cattle originally meant movable personal property. The first cow to appear in America arrived in 1611. (2) There are many different breeds of cattle, as well as classifications.

Bradford protein assay was conducted to evaluate protein concentration in the extracted liver. The presence of peptide bonds reflect the content of protein in

Protein A is thermostable, and is not destroyed by trypsin4; however, Protein A is most useful for its stability in regard to pH. It’s polypeptide structure is so stable that it has continuous stability both at very acidic pH (0.99) and at a very basic pH (11.8) 3,4. For this reason, it useful across many pH levels that would be useful in a protein affinity assay because this particular assay requires changing the pH to determine what would be the best to elute the protein that is being selected for; however, for an affinity assay involving human immunoglobulin, protein A is used for an additional reason. Protein A is from a bacteria and human immunoglobulin is associated with the immune system of the humans, as the name implies. Protein A contains five locations that it can bind to IgG5. These five domains, often labeled regions A-E, each consist of 58-62 amino acid residues, and a C-terminal consists of 150 amino acid residues. The IgG binding domains (A-E) each consist of three anti-parallel α-helices which are stabilized by hydrophobic regions between them. These domains bind with the Fc region of the immunoglobulin and form a complex with the Fc region. When this binding occurs a conformational change occurs which is reinforced by polar, and hydrophobic interactions4. This binding pattern is the basis for its use in protein affinity assay that involve eluting IgG out of solution that contain other human immunoglobulins. As the other

Two kinds types of microspheres modified with IgG (IgG-bead) and biotin-labeled IgG (Biotin-IgG-bead) were prepared as shown in Figure 4 (a). Two kinds types of microsphere, carboxylate-modified microspheres and orange fluorescence (540/560), were mixed in a carbonate buffer solution (0.1 M), and the mixture was centrifuged (1000 rpm, 5 min). After repeating the washing step more than twice, a small portion of PBS (10 mM, pH 7.0) was added to the microspheres mixtures of microspheres with the addition of carbodiimide. The reaction mixture was stirred for ˜∼180 min at room temperature. Then, the microspheres were washed twice with a PBS and suspended in