Bradford Assay

Bradford Protein Assay Practical Report 1. Present your data (including raw data and calculated concentrations) for the protein standards in the form of a clear table. Give one example of how you calculated protein concentration. Do not forget a descriptive title and units (4marks) Title either too long or not descriptive or absent Your results are in duplicate shouldn’t be referred to as ‘set1 set2’ or ‘original’ and ‘duplicate’ Many of you think units of absorbance are nm but A has arbitrary (ie no) units. nm indicates the ax of the chromophore Failure to give correct units in legends eg (ml) or (? g/ml) 2. Plot a graph of absorbance against protein concentration by hand. The graph should have an appropriate title and clearly labelled axes. Staple graph to the completed proforma and the Life Sciences submission sheet (4 marks) Mainly ok but both duplicate Abs- blank should be plotted and one line of best fit drawn through points. Do not extrapolate beyond the highest standard, you have no evidence that Beer-Lambert’s Law applies at high A. Make sure you choose appropriate scale and use full scale deflection on A4 graph paper. These types of graph are standard curves and that term should be in the title, remember we are not directly measuring the absorbance of protein, but a chromophore derived from the protein. 3. Present your data for unknown samples (including raw data and calculated concentrations of X Y) in the form of a clear table. Do not forget title and units. (4 marks) All data should be in one table but pay attention to typesetting and make sure that words/numbers are not split between 2 lines, this will lose marks. Absorbance of blank must be subtracted from values for unknown as they also contain non-specific absorbance. Many of you wrote dilutions incorrectly eg 1:2. The symbol : means ratio ? this actually means 1in 3. Either write as 1in 2 or 1:1 Never average absorbance-it’s not good practise (except for blank) you should convert to analyte then average your final results. Most dilute samples have least absorbance, many of you muddled your dilutions making final values incorrect. Always double-check arithmetic. If the final answer for the different dilutions don’t agree, look at your results and ask yourself if they seem right. Remember there is only one right answer for each unknown . Explain briefly each step of your calculations to find the protein concentration of X and Y, underlining your final answers. Convert to mg/ml. (6 marks) No need to explain how to read values from the std curve. Explain which absorbance values you read from the graph, what (if any) dilution factor you multiplied that value by, and then which answers you then averaged to get your final answers and why you ignored any data (eg poor duplicates or off scale cf standard- you cannot extrapolate beyond your std curve ) Some of you not using the proforma wrote too much. You will be penalised for exceeding allocated space in assignments, so be mindful of this 5. What is the chromophore measured in the Bradford assay? (2 marks) Many of you defined the term chromophore rather than describing the Bradford chromophore which is CBB + protein. (not CBB alone! ) The ? max at 595nm is formed when the dye binds to protein 6. What is the purpose of the blank? Why is it necessary to subtract the absorbance of the blank from all other results? (2 marks) The blank gives us the value for non-specific absorbance ; as we are interested in the specific (in this case protein) absorbance, we must subtract the blank absorbance from all other abs. values. Many of you didn’t subtract the blank from the unknown’s but as they are also mixtures of protein, NaCl and reagents measured in cuvettes, they also contain non-specific absorbance so you must subtract the blank. Many of you said the blank is used to zero the spectrophotometer (which it can be ) but we didn’t do that; we zeroed on water or NaCl then subtracted the blank mathematically. The blank you had to deduct was to remove the combined absorbances of water, NaCl and most importantly the dye in the uncomplexed state 7. The Biuret and Folin-Lowry are two other commonly used colourimetric protein assays. UV absorption can also be used to determine protein concentration. Describe the basis of these methods and compare them with the Bradford assay in terms of ease, sensitivity, range and interferences. (8 marks) You need to describe the biochemical basis(not the actual method) of the Biuret, Lowry and Bradford assays. The Lowry is a modification of the Biuret to improve it’s sensitivity so it’s appropriate to describe the Biuret method first , then describe the Lowry modification You need to state the range (the lowest- highest concentration they can detect) sensitivity(the lowest amount they can detect) for each assay. Some of you confused sensitivity with interference ie substances which, if present will give incorrect results. You need to state how reliable they are -whether they are prone to interferences. You could mention cost of reagents, ease of procedure Many of you placed too much emphasis on the ? ax of the different chromophores described but this is not really relevant. You need to state the wavelength at which proteins absorb UV radiation and which moieties in proteins absorb in the UV. ie at 280nm(near UV) it’s the aromatic amino acids, some of you also mention A200nm(far UV) at which peptide bonds absorb, although this is of little practical use. Note any inte rferences- remember many things absorb UV radiation Advantages of using UV- it’s non destructive so you can recover your sample for further investigation. Formula which relates UV absorption to protein concentration

The Priority for Glenmeadie's Innovation Efforts Essay

The Priority for Glenmeadie's Innovation Efforts - Essay Example Marketing experts should be aware that the product is the thing that they’re selling, and innovation in this field is also necessary to build a customer base. The purpose of this essay is to explore how Glenmeadie can balance their innovation efforts between the product and new types of marketing in an effort to impress new customers and build a loyal customer fan base, by using the Ansoff and 8Ps frameworks to provide an illustration on how best to tackle the issues at hand. Glenmeadie The information provided by Nunes & Driggs (2006) paints an outline of Glenmeadie. As a Scotch whiskey distillery, Glenmeadie has won 7 gold medals in one season on a national and international scale, suggesting an upmarket taste and therefore suggesting a target market of whiskey connoisseurs. The marketing campaign led by Bob consists of international efforts to put on Tastemakers events in 25 cities, starting in New York. The aim is to spend $15,000 on each event, offering tastes of various whiskeys and bringing in efforts from an apprentice distiller and buyers from local distributors. Glenmeadie is also trying to give a more personal face to the brand, having interactive websites and creating loyalty card programs. ... Rayport, Stephen Dull and Joe Scafido. Scafiado, a member of the executive council at Dunkin’ brands, raises concerns about the fact that the front- and back-house innovations programs seem to be presented as separate enterprise. This is mainly because they seem to have separate functions in marketing, with product development being considered part of back-room company enterprises and marketing focusing on customer involvement and sales. However, this is a limited perspective on the matter. At Dunkin’ brands, Scafiado brought together the culinary team and the operating specialists into one department, meaning that any culinary development is being innovated simultaneously with efforts on how to present this to the company. Herman, who is president of Lebanon, agrees that Glenmeadie should be focusing efforts on product excellence as well as drawing customers in with these innovative marketing campaigns. After all, it is the product that is being sold, not the marketing scheme. Rayport, founder and chairman of Marketspace, sees things differently. Although Glenmeadie has reported some stress on research and development budgets, Rayport suggests that this can only be a positive thing, even for Ellis, the distiller. Rayport even goes as far to suggest that Ellis’ argument against expansion in marketing efforts is a paradox, as Ellis cannot have the freedom to create and innovate in the distillery without an expanded market and expanded profits. Dull, vice president of strategy at Greensboro, sees things a little differently. Dull suggests that Glenmeadie is a luxury product, as a whiskey, and therefore the aims of the company to branch into mass-marketing are a mistake. Dull suggests that there hasn’t been much of an

The Importance of Self-Expression Essay Example | Topics and Well Written Essays - 1000 words

The Importance of Self-Expression - Essay Example This is what Charlotte Perkins Gilman explained in her literary work in 1892, â€Å"The Yellow Wallpaper†. With her own personal experiences on this predicament, she narrated her poignant tale of suffering, depression and nervousness. She asserted that vitality can be achieved only when pent-up emotions are given the right of self-determination. In her case, or of her narrator, suppression of emotions and lack of mental stimulation jeopardized her sanity and further worsened her mental illness rather than curing it. The monotony in her life turned out to be detrimental in her psychological well-being. As aforementioned, a mind needs an outlet to express the imagination it holds. This imagination is meant to be projected and communicated. Hence, a simple idea existing in mind can provoke a reaction and convey it in a coherent, lucid form. Be it in a form of art, words, plain gestures or even attitude, these expressions are a form or a medium for communication. If this connectio n is held back, only trouble brews inside the mind without any release or escape. Without release, mind and body, both go in a state of helplessness and lunacy. And this is what the narrator of â€Å"The Yellow Wallpaper† experienced. In her case, insanity took over because of her powerlessness to control her life. The constant reminder of not exhausting herself from any mental or physical work by her physician husband, John, situates her in a mental asylum. His patronizing attitude and lack of understanding about her feelings reduces their relationship into â€Å"...trust me as a physician?† (Gilman 278) one. Rather than interacting and understanding his wife’s problem, he abandons her to isolation of the house to cure her depression. Assuming that isolation from work and an idle mind would heal depression was actually not the right conjecture here. It’s because of this her imagination soars to new heights. With nothing worthy to do except stare at objec ts around her, she falls for the menacing yellow wallpaper in her bedroom. The horrid wallpaper then becomes her personal consolation and a subservient object of her undivided attention. She obsesses about discovering the intricate patterns and for the first time feels the thrill of the power to solve the enigmatic wallpaper patterns. The connection she feels after every discovery of the patterns is what excites her. This is how normally a sane person would react too. The need to express and share discoveries is ubiquitous and expressing one’s self or one’s perception is profound. Without self-expression, the communication process simply stops. The silence then becomes darkening and intimidating. And then eventually, the purpose of living easily succumbs to the black hole of nothingness. If this can disappoint a sane person, then the narrator of the â€Å"The Yellow Wallpaper† was already a victim of nervousness. The burgeoning desire to confront her problems pe rmits her to discover means of expression. But the restrictions imposed on any mental and physical work shun her mental capability. This provokes her to find reprieve in keeping a secret diary. By jotting her feelings, she finds a relation with the ‘dead paper and a great relief to my mind’ (Gilman 272). As she figures that her insight on any mundane task isn’t appreciated or understood by any of her family member, she resorts to writing. It becomes her sole companion who without any questions listened to her whine about her