Neutravidin Conjugation And Antibody Attachment 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 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

The results are organized in sections by order to which the experiments were performed. There are a total of four sections that is represented by a major heading and is shown in bold print that corresponds to the four experimental approached used in this study. Each section also contains either a table or a figure or a combination of both that corresponds to each experiment. The first section was to first figure out the best possible combination of these monoclonal antibody cocktails (ZmAb) and MB-003 by using laboratory animals most specifically guinea pigs and NHP’s (rhesus macaques) to test which combinations of MB-003 and ZmAb are the most effective and can be used for further experiments. To test the components of MB-003 (mAbs c13C6,

A horseradish peroxidase (HRP) is then added to each of the microtiter well which leads to the formation of a sandwich. The sandwich is composed of a polyclonal antibody, monoclonal antibody and chromogranin A. The unbound monoclonal antibody is later on washed out in the subsequent washing step. Detection of this immunocomplex is achieved by incubating the well with a substrate solution in a timed reaction and then a spectrophotometric microplate reader is used to take the readings (Stivanello

Neutrophils increase in response to a variety of conditions or disorders. In many cases, neutrophil levels increase as the body is trying to heal, such as with injury and trauma, or in fighting off an invading foreign substance. Acute infections, such as those caused by bacteria, viruses, fungi, and parasites can also cause an elevation in the number of neutrophils. Furthermore, an increased number in neutrophils can be seen in those with inflammatory disorders, such as the autoimmune disorder, rheumatoid arthritis. Some drugs, such as corticosteroids, can also cause an elevation in neutrophils.

As discussed, galectin-8 is has different functions such as cell growth and apoptosis. When galectin-8 interacts with different integrins, high affinity interactions are formed. When galectin-8 is soluble, it inhibits the cell cycle and prompts the arrest of cell growth [6]. This is caused by a 4-5-fold increase in p21, which is a cyclin-dependent kinase inhibitor [6]. An increase in c-Junc-n-terminal kinases (JNK) and protein kinase B (PKB) activities cause an increase in the levels of p21. When galectin-8 cDNA is transfected into cells of human lung carcinoma, the inhibition of colon formation is noticed; this suggests that when galectin-8 is overexpressed, it may inhibit cell growth [6]. An experiment was done to examine the effects of galectin-8. There were multiple procedures that took place in this experiment; the generation of antibodies against galectin-8, the creation of cell cultures with human carcinoma cells, generation of GFP-galectin-8, generation of GHO cells overexpressing a mutant form of SEK1, the preparation of cell extracts, the binding of galectin-8 to Lactosyl-Agarose beads, Thymidine incorporation, apoptosis, PCR, and a Northern Blot Analysis.

Two of the main functions of galectin-8 are cell adhesion and cell growth. These functions also occur in the protease-resistant form of galectin-8. Galectin-8 is a type of protein that is secreted. When it is secreted, is serves a modulator for cell adhesion [3]. When the protein is stopped, it promotes cell adhesion. This occurs by ligation and gathering of a select group of integrin receptors [3]. Recall that galectin-8 is a sugar binding protein. Therefore, when there is a formation between galectin-8 and integrins, it activates integrin-mediated signaling [3]. When there are large amounts of galectin-8 present, it negative regulates cell adhesion with it forms a complex with integrins. Cell adhesion depends on the interactions between galectin-8

Redness, swelling, pain, heat, and loss of function are all cardinal signs of inflammation. Acute inflammation lasts from a few minutes to several days, and is characterized by the exudation of fluid and plasma components and emigration of leukocytes, predominantly neutrophils, into the extravascular tissue. The physiologic mechanisms causing the wound changes are a result of the acute inflammatory response and the immediate vascular changes that occur, including vasodilation and increased capillary permeability; the influx of inflammatory cells such as neutrophils the effects of inflammatory mediators, which produce fever and other systemic signs and symptoms as seen in Carlton. Endothelial cells, platelets and neutrophils and Monocytes are

Undeniably, 2D monolayer cell culture method is one of the most popular models today in order to provide the first-hand information on the drug sensitivity on cell. In addition, this method is low costed and easy to be maintained [8]. But this culture method lacks the ability to mimic the tumour microenvironment such as the native extracellular matrix (ECM) of cancer cell [9]. In 2005, Zhang and his colleagues report that the cultured cells in monolayer can bring significantly difference in the rate of cell proliferation with those cell in vivo owing to the direct exposure to the oxygen and nutrient supply in monolayer culture [10]. Without the architecture of stroma, 2D monolayer model might provide inaccurate outcome due to the lack of true tumour heterogeneity in nature which consists of necrotic, quiescent and active cells [11-13]. Therefore, there is a necessity to adopt the three dimensional (3D) spheroids model first before conducting the xenograft studies as a prescreening assay to ensure a cost-effective, less time consuming and ethnically approved method in testing the sensitivity of the drug on cervical cancer cell [8].

I thought that it was interesting that we have cells within our bodies called neutrophils that are willing to self-destruct to save us from a potential infection. It is pretty cool how these white blood cells create their own little booby traps from their own DNA material. These traps are made to bind to Gram positive and Gram negative bacteria and inactivate them once a neutrophil.

Albumin, a 66.5 kDa protein, is quantitatively the most important plasma protein [85]. Albumin is the main determinant of plasma oncotic pressure, it plays a pivotal role in modulating the distribution of fluids between compartments, and exhibits many other biological functions, such as transport of endogenous and exogenous compounds, modulation of capillary permeability, neutrophil adhesion and activation, hemostasis and free radical scavenging (for review see 86]. Even mild oxidation of human serum albumin (HAS) impairs HSA functional properties including protease susceptibility, ligand-binding affinity and antioxidant activity [87]. The major structural change in oxidized HSA is a disulfide-bonded cysteine at the thiol of Cys34 of reduced HAS

Studies have shown that CN is a unique homodimeric disintegrin that is able to bind to many integrins. This ability gives CN an advantage among other disintegrins as they are only able to bind to a single integrin. Integrins are expressed in wound healing, tissue remodeling, angiogenesis, tumor growth, and metastasis (Schmitmeier et al. 2003). Furthermore, they are also ECM protein receptors, thus, they are involved in bidirectional signaling in the cell. As a result, integrins are able to modify a cell by altering migration, degradation, and proliferation among other functions (Swenson et al. 2004). Correspondingly, CN is heavily researched upon in order to combat illnesses. Disintegrins are able to bind to fibrinogen receptors, inhibiting fibrinogen platelet aggregation and decreasing tumor metastasis (Zhou

TRPC6, along with other relevant adhesion molecules such as PECAM and CD99, was found to localize strongly at junctional sites where there are transmigrating leukocytes. To answer whether this bears significance, independent experiments allowing polystyrene beads coated with relevant adhesion proteins to interact with an endothelial surface were conducted, finding that the ~35% of anti-PECAM coated beads specifically recruited TRPC6. In conditions where PECAM is blocked, TRPC6 activation, subsequent ↑[Ca2+]i,, and TEM was rescued by use of intermediates in the PECAM and TRPC6 signaling pathway. A generalized ↑[Ca2+]i induced by histamine failed to promote TEM, indicating that it is likely

Sections were incubated with the primary antibodies for 60 min. The antibodies used were galectin-1 (Genemed, clone NBP2,CA, USA, diluted at 1:400) and galectin-3 (Genemed, clone 9C4, CA, USA, diluted at 1:100).

More recently, a reverse micelle based polyacrylate coating method was undertaken to obtain various functional nanorods [24]. The micelle based coating was shown to be very versatile as the coating produced water soluble GNRs without particle aggregation. This coating developed could absorb a variety of functional groups including primary amines, ethylene glycol and fluorescein. This makes them much more suited for biomedical applications. This more recent study, although not highly cited does make progress in the GNR uses in biomedical applications and I believe should stimulate further investigation for this technique.