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Ouchterlony Double Diffusion Assay Biology Essay

Ouchterlony Double Diffusion Assay Biology Essay. Introduction: Polyclonal antibodies are produced by different B- lymphocytes in response to the same antigen, which recognise different parts of the antigen. Because the human immune system cannot know in advance what pathogens it will confront, it prepares for future infections by creating millions of different antibodies. Each of these highly selective proteins recognizes and binds to a specific target, or antigen, then signals other components of the immune system to destroy the target. These naturally-occurring polyclonal antibodies play a crucial role in triggering an immune response Polyclonal antibodies are routinely used as ligands for the preparation of immunoaffinity columns labeling reagents for the qualitative and quantitative determination of molecules in a variety of assays, such as double diffusion, radial immuno-diffusion, ammonium sulphate precipitation and ion exchange chromatography. Aim: The aim of this practical is to compare the purification of serum IgG by ammonium sulphate and ion exchange chromatography. The first purification step will normally involve a method such as fractional precipitation with increasing concentrations of ammonium sulphate. This method is not designed to achieve total purification, but to remove as much contaminant protein as possible whilst retaining all the protein of interest. Most proteins will precipitate from solution at high salt concentrations, but the salt concentration required to precipitate them varies considerably. Ammonium sulphate will be used as it is possible to set up salt concentration which will differentially precipitate serum proteins. Ammonium sulphate precipitation procedure was carried out to separate the serum proteins into four fractions. A fraction containing the serum protein to be purified can then be precipitated and collected, leaving behind any protein which is still soluble. The second method of purifying the IgG serum protein is ion exchange chromatography. This is a widely applied method of protein purification and uses positively charged groups or negatively charged groups immobilised onto a hydrophilic support, in this case DE- 52. Serum Proteins with an opposite net charge to that of the immobilised exchanger will bind to the column. Other serum proteins will pass through. Because the charge on proteins changes with pH, it is possible to attach a protein to the exchanger at one pH, then elute it by changing the buffer. Alternatively proteins can be eluted by passing an increasing concentration of salt through the column. The method works best for IgG which have high isoelectric points, at about pH 8.6. This method can also be used how to separate different subclasses of IgG. Ammonium sulphate is less effective in the purification if IgG, but it is useful for the isolation of large IgM. Samples of each fraction will then be separated by electrophoresis on an agarose gel. Antibody will then be allowed to diffuse towards the electrophoresed proteins from a trough cut parallel to the direction of electrophoresis. The proteins also diffuse from the positions they have reached after electrophoresis and precipitin arcs form where antigen and antibody reach equivalent concentrations. This technique can be used to determine whether a fraction contains any IgG and determine the degree of contamination of the IgG with other proteins Materials and Method: Ammonium Sulphate fractionation Procedure 0.25 ml of saturated ammonium was added to 1ml of human serum, to produce a solution which 20% saturated with respect to ammonium sulphate. The solution was mixed it was allowed to stand in an ice for 15 minutes, and was centrifuged for 15 minutes at 1500 rotation per minute. The supernatant was poured and pallet was retained as fraction 1 The supernatant from fraction 1; 0.35 was added to bring to 35 % saturated with respect to ammonium sulphate; the solution was left in an ice for 15minutes and it was centrifuged to recover the precipitate, the supernatant was poured in another tube while the pallet was retained as fraction 2 0.5 ml of saturated ammonium was added to the supernatant of fraction 2 to bring the solution to 50% , the solution was left in ice for 15 minutes to precipitate, it was centrifuged for 15 minutes the pallet was kept as fraction 3 while the supernatant containing 50% of protein was kept as fraction 4 the absorbance of the fraction was measured at 280nm the absorbance of 1mg/ml and 0.5mg of bovine serum albumin was measured was measured Before the immunological analysis the fraction salt content were reduced by dialysis against buffer. 0.2 (For more information refer to UEL hand out on protein purification) DE 52 – ion exchange chromatography Serum provides was pre dialysed against 10mM trs/barbitone buffer pH 8.6 and chromatography column containing about 2mls of DE 52 which it has been equilibrated in the same buffer The column was allowed to run until any overlying buffer has run into the DE-52 gel avoiding the column to dry Ouchterlony Double Diffusion Assay The fractions collected from ion exchange chromatography were determined for the presence of IgG by using ouchterlony double diffusion method. The collected fractions were run against an anti-IgG antibody in an agarose gel. The centre well were filled with 3ul of anti-IgG and 3ul of the eluted fractions into the surrounding holes. Immunodiffusion was slowed to proceed for 24-48 hours an antigen-antibody precipitin line was observed. Single Radial immune diffusion This as a quantitative technique whereby the antigen is allowed diffuse from a well into a gel which contained its specific antibody, a precipitin will form when antigen concentration is equal to the concentration of the antibody in the gel. Immunoelectrophoresis MATERIALS AND METHODS Preparation of antigen Blood samples were collected from ten clinically healthy cows using sterile disposable needles (1.2 – 40 mm), clarified by centrifugation (1000 g, 15 min) and diluted 1:1 with phosphate buffer saline (PBS, pH 7.2). Then equal volumes of diluted serum and saturated ammonium sulphate were mixed by slowly addition of the saturated ammonium sulphate solution with gentle stirring. After centrifugation (1000 g for 20 min), the precipitate was washed twice with 50% saturated ammonium sulphate solution. The final precipitate was dissolved in PBS followed by overnight dialysis against PBS. Protein concentration was quantified by a coomassie dye binding assay (Bradford, 1976), using bovine serum albumin (BSA) as the standard. Final protein concentration of solution adjusted to 1 mg/mL. Immunization of rabbits with bovine immunoglobulins Three hundred micro liters of prepared bovine immunoglobulins (1 mg/mL) in PBS was emulsified with equal volumes of Freund’s complete adjuvant (Sigma) and inoculated intramuscularly (I M) into three 6-month-old New Zealand White rabbits. The rabbits were fed regular commercial diets. The second and third inoculations were performed on days 21 and 35 with Freund’s incomplete adjuvant (Sigma), and the fourth inoculation was done on day 45 without any adjuvant. After the final immunization, blood samples were taken from the rabbits and production of antibody was investigated by double diffusion and ELISA tests. This study was approved by the Regional Medical Sciences Research Ethics Committee of Tabriz University of Medical Sciences. Purification of rabbit anti-bovine immunoglobulins Immunized rabbits sera were collected and precipitated by 50% ammonium sulfate. After dialysis against PBS and tris-Phosphate buffer (40 tris and 25 mM phosphate, pH 8.2), ion-exchange chromatography was done on a DEAE-Sepharose fast flow (Pharmacia) in a laboratory made column at a flow rate of 0.25 mL/min. Protein concentration adjusted to 100 mg/mL and passed through the column. The column was washed in two steps using Tris- Phosphate buffer for first washing step and Tris-phosphate buffer containing100 mM NaCl for second washing step. The eluted proteins were collected in 5 mL fractions and analyzed by SDSPAGE. SDS-PAGE analysis The purity of various IgG preparations was checked using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reduced conditions as described by Laemmli (Laemmli, 1970). The final concentration of polyacrylamide solution was 13%. Samples were boiled with 2% SDS for 10 min and were loaded on the electrophoresis gel. After separation, the proteins were stained with Coomassie Brilliant Blue G 250 (Blakesley and Boezi, 1977). Destaining was carried out in distilled water. Conjugation of rabbit IgG with peroxidase The conjugation was performed by the periodate method (Nakane and Kawaoi, 1974) with some modifications. First, 4 mg of peroxidase (Sigma) was dissolved in 0.5 mL of distilled water in darkglass container. Then sodium periodate (Merck) was added to the solution, and the container was kept on a stirrer for 20 min at room tempe-rature. The mixture was dialyzed against acetate buffer (0.1 mM, pH 4.4) at 4°C overnight followed by addition of 10 μl of carbonate-bicarbonate buffer (0.2 M, pH 9.5). Eight milligrams of purified IgG in 1 mL of carbonate-bicarbonate buffer (10 mM, pH 9.5) was add-ed to the active enzyme, and the container was put on the stirrer. Then 150 μl of fresh sodium borohydrate solution (Merck) was added to the above solution and was kept at 4°C for 1.5 h on the stirrer. The product was then dialyzed overnight against PBS at 4°C and 1% BSA (Sigma) along with addition of 0.01% sodium mirth-iolate (Merck). Enzyme linked immunosorbent assay (ELISA) Direct ELISA was used to determine the titer of HRP conjugated rabbit IgG against bovine immunoglobulins. 100 μl of prepared bovine, sheep and goat immunoglobulins, which was diluted 1:100 in PBS (10 μg), was added to each well of a 96-well micro titer plate and incubated at 4°C for 24 h. The wells were washed with PBSTween (0.05% Tween 20) three times and blocked with 200 μl of blocking solution (PBS-0.5% Tween 20). After a washing step, 100 μl of 1:400, 1:800, 1:1600, 1:3200, 1:6400 and 1:12800 dilutions of prepared HRP conjugated anti-bovine immunoglobulins were added to each well. The reaction was developed using 100 μl of 3, 3′, 5, 5′- tetramethylbenzidine (TMB) as substrate and the absorbance was determined at 450 nm after stopping the reaction by 5% sulfuric acid (Sigma). Results: RESULTS Production of rabbit anti-bovine immunoglobulins In order to survey production of antibody in rabbits and evaluating effectiveness of immunization, double diffusion and ELISA tests were performed. The titer of polyclonal anti-bovine IgG in double diffusion test was 8, which appeared as a sharp band between antigen and antibody wells. The titer of anti-bovine immunoglobulins determined by ELISA was 16000. Purification of rabbit anti-bovine immunoglobulins Purification of IgG rich fraction from immunized rabbit sera by ammonium sulfate precipitation followed by DEAE ion-exchange chromatography resulted in a highly pure fraction (first peak). The protein content of this fraction was 45 mg which was about one third of primary protein content (Figure 1). SDS-PAGE analysis Figure 2 shows the results of SDS-PAGE for determining the purity of IgG, which was purified by ion-exchange chromatography. A distinct polypeptide band with molecular weight about 50 kDa corresponding to rabbit IgG heavy chains. The diffused bands between molecular weights of 20 – 30 kDa correspond to rabbit IgG light chains. (Figure 2) The SDS-PAGE analysis showed that purification of IgG by ion-exchange chromatography resulted in a highly pure product. Discussion: The purification of immunoglobulins presents several practical complications, especially for polyclonal antibody production (Verdoliva et al., 2000). We used ionexchange chromatography for purification of rabbit IgG polyclonal antibody. Separation and recovery of proteins from ion exchange chromatographic media are affected by factors such as buffer type and pH, length of gradient, flow rate of the mobile phase, ionic strength and nature of counter ion, and characteristic of the proteins. The selection of ideal conditions for protein purification involves changing some or all of these parameters (Tishchenko et al., 1998). This technique was well established in our laboratory for purification of IgG antibody (Baradaran et al., 2006; Javanmard et al., 2005; Majidi et al., 2005). Furthermore, ion-exchange chromatography is considered as an economical alternative to affinity and immunoaffinity chromatography. After purification step we obtained a protein with approximate purity of 98%. SDS-PAGE analysis showed that the protein with approximately 50 kDa MW was rabbit IgG heavy chains. The light chain of rabbit IgG appeared as a diffused band of 20 – 30 kDa molecular weights. It is likely that diffused band of light chain could be related to different level of deglycosilation of protein during manipulation process. Conclusion: Ouchterlony Double Diffusion Assay Biology Essay
Social/Public Problem Paper. I need support with this Business question so I can learn better.

Social/Public Problem Paper: Each student will select a social problem. Students will then write a paper that discusses both the “social” perspective and about the “public” perspective of the problem (e.g. social problem: poverty public, problem: child poverty in America; social problem: gender inequality, public problem: discrimination of women in the workforce).
This paper should identify any particular characteristic of the population which suffers this problem or that is at risk. The paper should draw on considerable outside research and supply original and critical insights into the topic chosen. Your paper should only focus on the problem and not the public intervention.
Students are encouraged to use any but one consistent academic writing style. All papers should be 6 pages long and must be typed, Times New Roman, double spaced using 12-point font, and APA Format. Your paper must contain a minimum of 3-4 sources
Choose ONE of the following topics as your social problem for this paper:

Economic Disadvantage
Family & Community
Health and Medicine
Mental Illness
Physical & Substance Abuse
Crime & Delinquency

Social/Public Problem Paper

APU Risk Management and Its Importance Peer Responses.

Need a 250 word Peer Response with 1 cited reference Hello everyoneI hope everyone is having a great start to the week. This week we are talking about risk management. Risk management is the process of identifying, assessing, and controlling threats to an organization’s capital and earning. When assessing threats, these threats could come from a variety of sources. These threats could be to infrastructure, data-related risks, legal, natural disasters, and many more. Risk management identifies these and allows an organization to prioritize and take action against these. By implementing a risk management plan and considering the various potential risks or events before they occur, an organization can save money and protect their future. Within the current scope of my work, I deal with the Risk Management Framework (RMF) when it comes to Navy systems. As this is only one of many risk management strategies each organization will have the one that meets the needs of the organization. When applying the Risk Management Framework, this is done to mitigate any risks that are identified. This strategy is applied to system engineering as certain mitigation strategies require the addition of hardware. With additional hardware, systems have to take into account a wide variety of things such as the functionality of this hardware and how it is overall connected to the system. When it comes to software and overall security of the system, there are certain security configurations that need to be implemented. With these configurations, the system may not operate as intended. Due to this, system engineering needs to be involved to ensure that with the proper hardware, software, and security it is operating as it should be. Software updates and patches are always released and because of this, systems are evolving. System engineering is simply there to ensure that it is operating as it should be but also pushing the system to evolve with the advances in technology. I look forward to reading all the opinions and comments.V/rKurtisReferencesConducting a Risk Assessment. (n.d.). Retrieved August 24, 2020, from…Risk Assessment : OSH Answers. (2020, August 21). Retrieved August 24, 2020, from…Rouse, M. (2020, April 07). What is Risk Management and Why is it Important? Retrieved August 24, 2020, from…What Is Risk Management? (n.d.). Retrieved August 24, 2020, from a 250 word Peer Response #2 with 1 cited reference Class, Welcome to week 4! Half-way there and I hope everyone is doing well. Aww risk management and mitigation. As I am sure anyone who has any sort exposure to the military can attest to this is a very common concept. It even has its department directive (DD Form 2977, Deliberate Risk Assessment Worksheet). This form lays out the risks associated with whatever activity we are applying for so that the command team can approve or disapprove the form. We even have to use it for situations like washing vehicles (Department of Defense, 2014). The reason that this form is even in existence, is for legal reasons because someone at some point got hurt or damaged something in whichever activity they are doing. Anyway I suppose this is the purpose of risk management. Learning from past mistakes to help determine is the risk is worth the activity itself.When it comes to information systems Jacobs states that risk management is “the process of reducing the risk faced by the enterprise, through risk mitigation actions and risk assignment agreements, to acceptable level of residual risk that the organization can consider a normal cost of doing business” (Jacobs, 2016, pp 224). In laymen’s terms risk assessments in the business world are associated with the lowering of costs. A perfect example of this in my mind is when large companies factor in the cost of a law suite when dumping toxic waste. Basically they performed a risk assessment and found that the cost disposing of waste in a safe and legal way outlays the cost of even the harshest law suite. Its simple dollars and cents. This is a rather cold examination of risk management, but it makes since from a purely business perspective. In information systems risk management, boils down to how much risk an organization is willing to take on with regard to there information. For example if a company decides to get into health insurance they need to weigh how much it will cost to protect data in compliance with HIPPA. Now when dealing with data in particular there are other risks outside of financial costs. These include a lack of trust in the organization/data, a loss of contracts to handle the data, and in extreme examples jail time for top executives. When building risk tolerance into an information system our textbook states that the risk management mythological framework revolves around “identifying risks and determining how to manage (reduce) those risks” (Jacobs, 2016, pp 232). Both of these two goals must be met enable for the risk tolerance to be within the system. Much like how on the DD Form 2977, it is impossible for the system engineers to fully know every risk they need to base there assessment on historical analysis. A key factor in these systems is building enough flexibility into the system so that once it is cracked open by attackers they can be quickly quarantined and the system is updated to reflect the new risk. Anyway I got a little off topic throughout this weeks post, but I look forward to hearing everyones thoughts. As always if anyone needs anything from me, please just reach out. References Department of Defense. (2014, January). DELIBERATE RISK ASSESSMENT WORKSHEET. Retrieved August 24, 2020, from…Jacobs, S. (2016). Chapter 4-5 In Engineering information security: The application of systems engineering concepts to achieve information assurance (Second ed., pp. 123-267). Hobokin, NJ: John Wiley & Sons.
APU Risk Management and Its Importance Peer Responses

SAS Statistical Software Needed – Data Management & Epidemiologic Analysis

SAS Statistical Software Needed – Data Management & Epidemiologic Analysis. I need help with a Health & Medical question. All explanations and answers will be used to help me learn.

Exercise 1.2 on page 20 (SAS textbook)
Consider the following data set (FHEALTH) representing measurements on female’s health data. Using SAS statistical software, perform the following

Import the data into SAS.
Draw histogram for the variable age.
Draw a box plot for the variables “BMI” and “Pulse”, what is the shape of each variable.
Calculate the descriptive statistics (mean, median, standard deviation, Skewness) for the variables Pulse and BMI.
What is the shape of the variable Weight (wt)? Illustrate histogram or box-whisker plot.

SAS Statistical Software Needed – Data Management & Epidemiologic Analysis

HIST 100 SDCC Heroic Qualities & Flaws of Major & Principal Characters Discussion

online dissertation writing HIST 100 SDCC Heroic Qualities & Flaws of Major & Principal Characters Discussion.

Instructions:Write a two- to four-page paper on one of the questions below. The paper should follow MLA or University of Chicago format and include proper citations and appropriate examples. A successful paper will include at least six specific examples. Your paper should have a title. Additional research is not necessary, though you may wish to use one or more of the articles in the Norton edition of the book.Choose one and only one of the following options:What lessons does Gilgamesh learn about seeking fame and then eternal life?Discuss the relationship between humans, semi-humans, and the Mesopotamian gods. What does the epic tell us about the nature of Mesopotamian religion? Who are the important women in the epic and what role do they play in the story? What do the portrayals of women tell us about how women were seen in ancient Mesopotamia?What heroic qualities do the major characters possess? What flaws do the major characters have? Explain.How to include Proper Citations:Since this is a one-source paper, you need only to cite a page number when you reference examples. Be sure to include a citation for a direct quotation AND for a general reference to something in the book. Example 1: Enkidu warned Gilgamesh that the “hunt of Humbaba is a hopeless quest.” (18)..If you use additional sources, such as one of the articles in the criticism section, you will need to cite these too. Again, you need to include a citation whether you are quoting or if you are making a general reference.Example 1: Rivkah Harris points out that women “are regarded positively only when they assist Gilgamesh….” (Rivkah Harris, “Images of Women in the Gilgamesh Epic,” 209.) Example 2: Only women that help Gilgamesh come across positively. (Rivkah Harris, “ Images of Women in the Gilgamesh Epic,” 209.)In example two, I borrowed the idea from Harris, so I need to cite her work, even though I am not quoting her directly.Dictionaries and Encyclopedias: It is considered poor style to quote the dictionary or an Encyclopedia. These sources are used for background information. Under no circumstances should Wikipedia be used. Any paper making reference to Wikipedia will not be graded.
HIST 100 SDCC Heroic Qualities & Flaws of Major & Principal Characters Discussion

Woodards Theory of Cultural Geography Theoretical Analysis Essay

Woodards Theory of Cultural Geography Theoretical Analysis Essay.

Hi, Class!OK. Time for the FINAL ESSAY, due any time after Thanksgiving and before the end of the semester. It is intended to be a 5-10 page (double spaced, 12 point, Times New Roman) analysis of the accuracy of Woodard’s theory of Cultural Geography in our text, “American Nations”, as you have experienced it or imagined it to be, in one specific location from one of Woodard’s “nations.” Of course, let me explain:Let’s say you are from Hoboken, New Jersey, or want to be; or know somebody who is, or have a relative there…or whatever. Woodard says this is New Netherland and it was characterized by Dutch ethics and culture, which, he says, are still retained. Your family and town – the O’Briens of Hoboken – let’s say – is supposed to be tolerant, interested mostly in making a buck; doesn’t mind learning other languages as long as they can make a profit…etc. (according to Woodard.) Analyze and agree or disagree. (Obviously, there is no “right” answer here; you can disagree with Woodard if you want, or you can hate him or worship him. I don’t care.).Next, research the history of Hoboken (or the Oranges, or what ever) and the O’Briens and compare what you find to Woodard’s rendition. Then, describe Hoboken as it was and as how it is now, the demographics, the sources of income, etc. and, if you can, the cultural “vibe” (Eg. Every weekend there are tag sales on every lawn. Lottery scratch offs sell like hot cakes; anyone who wants to can join the AOH as long as they have the money; and the bar is open to anyone, regardless of race, creed, gender, etc.) . Make it personal wherever possible (Eg. “My grandfather used to say, ‘You should be eating like a Dutchman!'”). And then – determine the accuracy of Woodard’s theory regarding Hoboken and New Netherland, it’s history, its ethics, and the extent to which his theory is correct (or not) and whether (or not) you can still see the Nation of New Netherland in operation, even in your family.FINALLY, come to some conclusions as to the usefulness of Woodard’s version of Cultural Geography as it applies to your topic (Hoboken, New Jersey), and also come to some conclusions as to the usefulness (or lack of usefulness) of the concept of Cultural Geography in understanding the ethics, behavior, political decisions, etc. of America (You may argue that the whole concept is divisive, inaccurate, and misleading. I don’t care; but you must back it up with the same detailed examples from current or past history as you would have used to prove that the concept is absolutely correct).NOTE AND REMINDER: Keep rereading this question before, during, and after responding. IT IS NOT ABOUT THE HISTORY OF HOBOKEN, NEW JERSEY. PLEASE DO NOT HARRASS THE O’BRIENS OF HOBOKEN IF THEY ARE NOT YOUR RELATIVES! OU DON’T HAVE TO PICK NEW NETHERLAND! YOU DON’T EVEN HAVE TO CHOOSE THE TOWN AND REGION IN WHICH YOU WERE RAISED. THE ABOVE IS JUST AN EXAMPLE.ALSO, HAVE FUN WITH THIS! This theory is relatively new; what you write about probably hasn’t been written about before by anyone; and your paper could easily be revised a little and submitted to a regional magazine, a newspaper Feature section, or even a scholarly journal!
Woodards Theory of Cultural Geography Theoretical Analysis Essay

Theories of International Trade and Investment

Theories of International Trade and Investment. I need an explanation for this Business question to help me study.

Explain how nations can enhance their competitive advantage. What are the determinants of national competitiveness? Provide at least one example of a country with successful national industrial policies.Your well-written paper should meet the following requirements:

Be 4-5 pages in length, which does not include the title page, abstract, or required reference page, which are never a part of the content minimum requirements.
Use APA style guidelines.
Support your submission with course material concepts, principles and theories from the textbook and at least two scholarly, peer-reviewed journal articles.
cover all the requirment

Theories of International Trade and Investment

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