Scientific Report Expectations for Determination of Protein Concentration by Lowry Method

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it is a formal report, and all the information needed is uploaded in the file (one with all the information needed, the other with all the results needed)

– in the Excel file you will find the graph that needed to be put in the formal report

-what needs to be done:

•  You must pay attention to grammar and spelling.
•  All statements must be in indirect speech and do not use first or second person. (No, “I’, “we’ “us”,
  “they, etc”).
•  For example: Instead of stating “I used Visible spectrophotometry”, you should state it as “Visible
  spectrophotometry technique was utilized”
• 1. Cover Page: Use the cover page template document posted in Blackboard, and complete the rest
     of the report in that document as per the following sections:
  2. Abstract: This is an overall summary of the experiment, the results you got and your
     conclusions. Write a paragraph, briefly in about 100-150 words total, include the following:
  3. Peterson, Gary L. “Review of the Folin phenol protein quantitation method of Lowry, Rosebrough, Farr
     and Randall.” Analytical biochemistry 100.2 (1979): 201-220.
     Sapan, Christine V., Roger L. Lundblad, and Nicholas C. Price. “Colorimetric protein assay
     techniques.” Biotechnology and applied Biochemistry 29.2 (1999): 99-108.
     Waterborg, Jakob H. “The Lowry method for protein quantitation.” The protein protocols handbook.
     Humana press, 2002. 7-9.
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     which protein you worked on
     what was the aim of this specific experiment
     which experimental method and instrumentation technique were utilized
     important result(s), and
     your major conclusion(s).
     Introduction: Address the following aspects within 500 words..
     •  Why is it important to determine protein concentration? (what are some of its applications?)
     •  A brief introduction and description of the principles of spectrophotometry in general.
     •  Which amino acids in a protein can give UV absorption at roughly 280nm?
     •  Why can’t the UV absorption be used for protein determination? Why is a color-producing
       reaction necessary?
     •  Objectives or purpose of the experiment.
     •  Which reaction method? Give Details of the two-step reaction/mechanism
     •  The advantages of using the two reagents.
     •  Sum up in one statement, what you set forth to do in this experiment and using which protein.
       Experimental Materials and Methods:
       Under Materials
     •  Describe the different reagents that were used, the different equipment and instrumentation that
       were used. Be specific to the ones that were used in the lab, and you can include pictures.
     •  What was the Unknown number that was given to you?
     •  Hazardous nature of reagents and proper waste disposal.
       Under Methods:

     o You can add any pictures you may have taken during your experiment. But make sure to give figure
     numbers and title to the pictures.
     o Briefly and succinctly, describe the procedure steps you used, in your words (do not copy and paste
     from the procedure posted by the instructor), from preparation of standards, the reaction steps and
     to final measurements of absorbance.
     o Describe what type of blank was used for calibration of the Spectrovis
     o Mention the wavelength of maximum absorbance used and the pathlength of the cuvettes used.
     o Show examples of calculation of how the standards were made. Include Table-1
     o Be sure to specify the quantities of materials used and their units.
     o Data Analysis Method that was used.
     Results:
     You can add any pictures you may have taken during your experiment. But make sure to give
     figure numbers and title to the pictures.

     •  Describe your visual observations of color and intensity of color you saw for comparisons of blank
       and the different standards.
     •  Did your absorbance data at 750nm, compare with your visual observations for your standards
       based on their concentrations?
     •  Include the Data Tables for standards preparation and the Table of absorbance results.
     •  Include the graph you plotted, making sure it has the correct title, axes labelled, all units shown,
       and the linear regression and R2 value displayed on the graph.
     •  Absorbance values obtained for your unknown.
       Calculations for unknown:
     •  Show calculations of the unknown concentration (for each measurement you did in duplicate)
       using the linear regression equation from Excel
     •  Calculate the “mean” of the above results.
     •  Carry out another calculation for both of your unknown duplicates, using the formula shown
       below, by using the Standard which showed absorbance nearest to the unknown:
       Concentrationofunknown=AbsofUnKnown
       Abs Standard
     •  Calculate the “mean” of the above results
     • • Calculate the percent difference between the mean value you got from using the linear regression
         equation, vs. mean value from comparison to the nearest standard.
         Percent difference =
         (Mean Conc. from Std Comparison – Mean Conc from Calibration Curve ) x 100
         (Mean Conc. From Calibration curve)
       •  What was the “true value” of the unknown (based on information obtained by the instructor)?
       •  Now, Calculate the percent difference between what you obtained from Excel linear regression
         curve and the value given by your instructor.
       •  Percent difference = (Conc. from Calibration Curve – True value ) x 100
         True Value
         Discussion:
         Some suggestions are given below. You can add any other things you find to be important.
         (make sure it is all written in essay format).
       •  What were your initial expectations with regard to Lowry method in your particular experiment?
         Did the reaction work the way you expected and produce the color expected?
       •  Did your results of absorbance of the different standards obey Beer-Lambert law? How do you
         know this?
       •  How did your R2 value compare to theoretical value? What are your conclusions about this?
       •  How did the Mean concentration of the unknown calculated based on nearest standard
         absorbance, compare with the Mean concentration based on linear regression equation from the
         Calibration Curve?
       •  Which one is a more accurate method of calculating among the above two ways, and why?
       •  How did the Mean concentration of the unknown determined from the Linear Regression
         equation match with the “true value” of concentration given to you by your instructor, in terms
         of percent difference?
       •  The percent difference allowed in this experiment is 10%. Comment on the percent difference
         you obtained.
       •  What are the different possibilities of errors that could have occurred during the experiment if a
         student does not get close to expected value within allowed error %. (do not state is as errors in
         devices and chemicals/reagents! All devices in the lab have been tested and proven for accuracy!)
       •  Is Lowry method the only method that exists for protein estimation? If not, which are the other
         methods available, and what is the reaction they are based on?
       •  When several methods are available, how could one go about choosing the most appropriate
         method for the type of sample?
       •  What are the advantages of Lowry method?
       •  What are the disadvantages of Lowry method?
       •  What are some of the interfering substances in Lowry method? If you had such interfering
         substances in your sample to be analyzed, what would you do?
       • Can Lowry method be used for estimation of free amino acids not bonded as peptides as proteins?
         If not why not?
       •  Can Lowry method be used for estimation of proteins/peptides from different samples such as
         cell-extracts, soil extracts, food samples, etc? If yes why, if not why not?
       •  Look up the nutritional value in terms of protein on any food sample you have at home (any
         packaged or processed food you have at home, or milk or yogurt containers you have). What is
         the amount of protein per serving they have labelled on it?
       • •  Now, how do you think that manufacturer determined this protein content? You can make
           assumptions based on the theory you have read so far about all the different methods of protein
           concentration determination. Or you can look up on the internet, specifically for food related
           determination, and describe.
           References
           List all references that were utilized in your report, in the APA style.
           Examples of reference styles acceptable in chemistry or biochemistry. These are just examples,
           not applicable to your experiment; they are provided just to show how the references should be
           written.
           (Use only one style in any report.)
           For books
           Garrett-Goodyear, J., Harries, J., Patey, D., Shook, M. Writing Papers: A Handbook for Students
           at Smith College, 2nd Ed.. Littleton, MA: Sundance Publishers Inc., 1986.
           Clark, J. M., and Switzer, R. L. 1977. Experimental Biochemistry. San Francisco: W. H. Freeman
           and Company.
           For scientific journal papers
           Clark, J. & Nicklas, W. 1970. “The metabolism of rat brain mitochondrial preparations.”
           J.Biol.Chem. 245: 4724 – 4731.
           For internet sources
           Name of the author(s). Topic. Available from: name of website. [year, date of information taken]
           Groot, M. N. Conversion of phenylalanine to benzaldehyde. Available from:
           Hhttp://www.ftns.wau.nl/imb/research/phenylalanine.htmlH. [2001, Oct 2]
           The NutraSweet company. Aspartame sheet fact. Available from:
           http://www.nutrasweet.com H [2006, Dec 15]