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Guide to the study

• membrane organization
  
• overview of the study
• research paper

Lab part 1

• components of blood
• blood and red cell fractionation

Lab part 2

• introduction to SDS-PAGE
• preparing gels
• preparing samples
• assembly, loading, running gels

Lab part 3

• gel images
• gel analysis
  
• molecular mass standard curve
• measuring relative mobility
• "Hall of Shame"

Selected methods

• tissue fractionation
• centrifugation
• Bradford protein assay

Research Paper – Characterization of Erythrocyte Cytoskeletal Proteins by SDS-PAGE

When you complete the entire characterization you will write up the study in a form that is used by most journals in biosciences. Before you even start the laboratory work please look over the introduction and recommendations for the individual sections. Doing so will help you understand the context of the study. Knowing in advance what you will need for the write-up should help you to make sure that you obtain and record all of the necesary information. This paper will require data from blood and erythrocyte fractionation as well as from analysis of protein gels.

Honor code policy

You may work together with your lab partner(s) in acquiring data from your protein gels, and may work together in making identifications of protein bands. You may analyze one or more gels that were prepared by other students, provided that you acknowledge the source of the data and that you include your own gels in your paper. Any collaboration is limited to the acquisition of information. All writing, preparation and labeling of gel figures, preparation of tables, etc. is to be done individually.

Introduction to the assignment

Technical writing may seem overly demanding to the beginner, but adherence to the "rules" is critical to the precise communication of facts and concepts in the scientific community. To prepare your research paper you must consult the recommended web pages, examples, and resource materials. An editor will require that you adhere to a specific format when you submit a paper. The same policy applies to this course.

For the first paper, very specific guidelines are provided. Focus on clear, concise, well-organized writing, appropriate style for each part of a paper (and why), careful analysis and careful reporting of quantitative data, and reasonable interpretation of the results. Be prepared to apply what you have learned here to the next paper. The guidelines next time will not be as specific.

Specific Recommendations

Title page

McMillan (4th ed.) p. 69-72 discusses choosing a title for a paper, illustrating the text with quite a few examples. You can quickly draw the conclusion that "Biology lab #1" is really not an effective title. The title should concisely describe the focus of the paper. What is the primary goal of the study? See if you can write a title that concisely sums up that goal.

Materials and methods

Before starting on this first writing assignment please go to the Policies page and read up about how to submit papers. See McMillan (4th ed.) pages 78-83 for advice and examples. Give some attention to the discussion of the difference between active and passive voice (page 82). The general guidelines for writing research papers are presented in writing/analytical resources section. You might look over the entire document to get an idea of the general organization of a research paper and specific recommendations for a materials/methods section.

In Materials and Methods we document the methodology that we used to accomplish our goals. This section will be a lot shorter than you think it should be. We include only the barest essentials, omitting any unnecessary information. After all, the focus of the paper should be on the results and their interpretation, not on the methodology itself. This section will be a lot shorter than you think it should be.The purpose is to permit a reader to judge the scientific merit of the work and/or to reproduce all or some of the methods. In the latter case the reader will apply the methods to his/her own study, not yours, so report a general methodology, not details that were specific to your experiment.

To help you get started, part of this first assignment has been completed for you. It is your responsibility to complete the section. Notice the general style and content of this example and of the other examples provided to you in the writing example package and writing text. Notice that we do not use informal lists. We write in normal prose, using complete sentences.

Because you are documenting specific methods that you applied to this completed study, past tense is appropriate. As for voice, the choice is yours. To use active voice you almost have to write in first person. After all, a beaker doesn't fill itself. Because the reader's attention should be directed to the subject matter itself rather than to the author, many authors use the passive voice to avoid having to use first person. Often, a passage reads more smoothly in the passive voice, but if passive doesn't work you may use first person. The important thing is to communicate the information effectively.

Use the following section as a template for the Materials and Methods for your first paper. Your job is to write up the fractionation procedure, starting with receiving the whole blood sample. Methodology related to protein assay and SDS-PAGE is complete. You may change the style of the second part to be consistent with the part that you write, but don't add anything.

Here are more suggestions for what NOT to do.

• Do not tell a story. The chronological sequence of methods is implicit in the writing, so you need not write that first you did this, then you did that, etc.
• Do not report using nonspecialized supplies and equipment such as pipettes, glassware, inorganic chemicals, spectrophotometers, or electrophoresis supplies; such use is implicit in the procedures and need not be specified in methods.
• Do not report details that are irrelevant to a third party, such as what knob to turn on the microscope or what day the experiment was conducted.
• Do not refer to the teaching lab, instructor, students, team, etc. This is not a "lab report." Published research articles only list authors under the title and acknowledge additional assistance at the end of the text. They treat the study on its own merits. Who did what is not relevant.
• Do not deviate from past tense.
• Do not list materials or provide outlines. Stick strictly to prose.
• Do not provide explanations, definitions, or background of any kind.

Materials and Methods

Fractionation

***It is up to you to write up this part. Write up the methodology only, as none of the materials is specialized so as to require noting a source. Try to be concise without sacrificing clarity and accuracy. Please do not turn in the section on assay and characterization that I wrote for you below. Just indicate where in your materials and methods section it should be inserted.***

Assay and Characterization

Bovine serum albumin (BSA), dyes, acrylamide, dithiotheitol, and molecular weight standards were purchased from the Sigma Chemical Co., St. Louis, MO.

A Bradford assay (Bradford, 1976) using BSA as standard was used to estimate protein concentrations. Aliquots were stored frozen without denaturation for up to a week.Samples were denatured within an hour of electrophoresis by diluting to 2 mg/ml protein in 1% sodium dodecyl sulfate, 10% glycerol, 1 mM ethylenediaminetetraacetic acid, 160 mM dithiothreitol, 0.1 mg/ml bromophenol blue dye, and 10 mM Tris-Cl, pH 6.8, followed by heating at 80C for 10 min.

Electrophoresis was conducted by the method of Laemmli (1970) on 3 1/4 x 4" gels prepared from 30%T, 2.5%Cbis stock. Gels were stained with 0.1% Coomassie blue dye in 50% methanol, 10% glacial acetic acid.

Would be included in Literature Cited

Bradford, MM. A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254. 1976.
Laemmli, UK.  Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:  680-685.  1970.

Results

Please examine pages 83-89 of McMillan (4th ed.). Summarize your findings in text, and illustrate your results with an appropriate figure and table. The results section is a writeup, first and foremost. An effective writeup describes the major findings in the context of the objectives of the study. For example, for a study on the effects of a new drug on blood pressure one might write, "To determine if the testing process itself raised the subjects' blood pressure, ten subjects were treated with a placebo. A paired t test revealed no significant change in blood pressure within this control group following treatment (Table 1)." Recall that your results are history (past tense), and they are certainly not generally accepted truths, as would be implied by the use of present tense.

Rather than write a bare minimum of text, think of useful observations that you might add. For example, are any of the observations that you made during the centrifuge runs worth noting? Were there any difficulties or complications that might compromise one's attempt to reproduce this work? How many well-resolved bands showed up in the membrane fraction of lower density gel and what MW range was separated? How many additional bands were resolved by the higher density gel (beyond the range of the first gel)?

Your writeup should certainly describe the major findings that are illustrated by figures and tables. Your text should complement the figure and the table rather than serve as an alternative source of information. You might report quantitative information that is not directly revealed by figures and/or tables, point out the most important findings, and/or draw direct conclusions from the data. There is no point in repeating the same quantitative information that is presented in the table.

Do not include interpretation in the form of explanations, and do not include background information. Only the results of the current study should be presented here, that is, just report the facts.

Results to include

Manuscripts require that figure and tables be prepared in publishable form. You don't have the latitude that you might have when preparing a talk or poster. See chapter three of McMillan, "Using Tables and Figures." Pay particular attention to numbering, and content/placement of captions and titles, and to the use of separator lines in tables. Keep all parts of a figure or table on the same page, using a separate page if you find it awkward to incorporate the item into the text.

All measured quantities or derived values that are based on measurements are uncertain quantities. As with the abstract, or in ANY part of any type of communication, report ALL uncertain quantities to an appropriate degree of precision. This includes quantities reported anywhere, including the results and abstract of a paper.

Fraction yields. It may be valuable for an investigator to know how much of a specific protein or mix of proteins can be obtained from a given amount of whole blood, using your fractionation procedure. Perhaps he/she wants to study hemoglobin, and wants to know how much can be obtained from your fraction 3. See the web page on tissue fractionation for the concepts of protein yields and how to obtain and report them. It is customary to report yields in a table with (for each fraction) name of fraction, protein concentration, volume of the fraction, and total protein.

The gels themselves are your most important result, of course, and since the paper focuses on analysis from the gels you should include reproductions of the gels that you analyzed. They must be neatly and properly labeled, and numbered with captions. Photos, graphs, drawings, etc. are simply figures (figure 1, 2, etc.) and are to be presented as figures in a paper. Figures should be capable of standing alone. For example, when you refer to a protein band in the discussion, there should be no question as to which gel photo shows the band, where it is in the photo, with what fraction it is associated, and what percent gel resolved it.

As a basis for discussion, you will need to describe the bands. The study focuses on membrane associated bands, of course, although the disibution of hemoglobin should also be discussed. Distinguishing features of protein bands include: fraction with which the band associated, apparent MW, intensity of staining, unusual associations such as being part of a doublet (two bands close together of near equal staining intensity), unusual shape. There will be a lot of data here. While reading the discussion, a reader will want to be able to access specific information quickly. Therefore such data are best represented in one or more tables. Remember to report only pertinent data, not intermediate calculations.

Discussion (no more than five pages)

In addition to the materials provided during the analysis session, you may wish to make use of the information on structure of the red cell membrane posted on this web site. An article on membrane proteins has been posted as well. It is essential that you use the information in the article on the organization of the red cell membrane. A cell biology text might also make a useful general reference. See McMillan (4th ed.) p. 89-94. Pay particular attention to the very first bulleted point. Make an outline of what you plan to discuss. A suitable outline might go like this.

• Comments on the fractionation and yields
• How well was each fraction separated? Was the isolation of membranes successful and what is the basis for your conclusion?
• Major proteins that were resolved; identities and supporting evidence/reasoning to support your conclusions; to effectively defend your conclusions you must incorporate functions of the candidate proteins into your reasoning
• Bands of questionable identity, possible alternative identifications
• Known proteins that could not be postitively identified
• Distribution of hemoglobin including where found and presence of multiple bands

This is just one of many outlines that might work. Make whatever modifications you feel are appropriate, as long as you include all of the pertinent interpretation. Discussions are evaluated mainly on the basis of completeness, precision and accuracy of language, and depth of interpretation. For all conclusions that you draw or suggestions that you make, provide your reasoning and support your reasoning with evidence from the laboratory and/or from the literature.

Abstract

An abstract is a concise summary of the major points of the paper. Many times a reader will read the abstract to a paper and not go any farther, so it is necessary to pack the most important information into a relatively short passage. Most abstracts are written as single paragraphs, and that is how they are to be submitted for this course. See McMillan (4th ed.) p. 72-76 for guidelines and examples.

What was the objective of the study? You should be able to summarize the objective in a single sentence. It is not necessary to defend the objective or provide any additional background. The focus is to be on the results and conclusions. As suggested by McMillan, a good abstract uses very specific language. We do not dwell on specific methodology, although the general approach to the problem should be summarized, again very briefly. See the examples of abstracts that are too general and too wordy, respectively, in the McMillan text. Summarize the results of the entire study, completely and accurately, in both qualitative and quantitative terms. Don't forget to express quantities with appropriate precision.

A trick for being concise is to incorporate two bits of information together. For example, "to reproducibly separate and identify proteins of the coat of bacteriophage T4, phage proteins were denatured in an SDS buffer and separated on discontinuous polyacrylamide gels." One sentence sums up the objective and the general method, allowing the writer to focus the rest of the abstract on the findings and conclusions. ***NOTE*** The single most common mistake in student abstracts is to omit specific findings. Reporting findings is the most important purpose of an abstract. In particular, students frequently omit quantitative information (mean values, errors, statistics, etc. for key findings).

Introduction (maximum two pages)

So why write the introduction last? Many students have commented that they really didn't understand the full context of the study until after they wrote up the discussion. We'll try it this way and see what happens.

Describe and defend the study, so that the rest of the paper makes sense to the reader. See McMillan p. 76-78 for guidelines and examples. .

• The overall plan is to describe and defend the overall goals of this area of research, the specific objectives of this experiment, how the experiment was designed, and how it accomplished its objectives.
• Provide factual information only to make a point. There is no need to tell the reader everything you know about blood, for example.
• Provide a context. What is the relationship of this study to our overall research?
• Why did we choose this particular experimental model? There were practical reasons for using this starting material.
• What, precisely, was the experimental objective?
• Describe how the experimental design accomplished its objective, without providing excessive detail on the methodology itself.

Some authors use future tense in an introduction, but since the work will have been completed at the time the paper is completed, past tense is most appropriate. Of course, we reserve present tense strictly for established facts and concepts, and do not refer to specific results, conclusions, decisions, past events, etc. using present tense.