Introduction: (1 page, 10 pts). Do not paraphrase the 1st page of this handout.
A. Introduction to background information about erythrocytes in general and membrane structure, composition, and the fluid mosaic model
B. Experimental goals of the lab.
C. What use, clinical or research, could the information from these types of experiments be put to?
D. Brief description of the general procedural approach used for each goal.
II. Materials and methods: (2 pages, 5 pts)
Give a BRIEF synopsis in paragraph form of just the essential concepts of each procedure. Explain it as if you were trying to explain what you did to a scientist who already understands the techniques involved. Do not give a narrative of how the procedure was performed or a numbered list of the steps: DO NOT just retype the lab handout procedure. Include important specific information: blood cells, reagents, centrifuge g-forces and times, and what calculations were performed. Also, include any errors or changes to the procedures.
As briefly as possible, recount the methods used to:
i. Isolate Membrane
ii. determine surface carbohydrate (Hemagglutination assay)
iii. determine Lipid types (TLC)
iv. determine membrane protein asymmetry and types (labeling and gel)
v. lipid/protein ratio (gravimetric analysis and Lowry)
III. Results: (10 pts)
This section should contain two parts for each experimental goal:
1. A rationale and experimental approach. Usually, the rationale and approach are one or two sentences. The results should follow a rationale for the relevant experiments (e.g. how does the labeling tell you about protein asymmetry?).
2. A presentation of the results, including a summary of the important points. The results are often presented as a table, figure or photo and are summarized in a sentence or two. You should present the results from these experiments:
A. Cell surface carbohydrates (agglutination assay)
B. Types of Lipids Present (from TLC)
C. Protein to lipid ratio (Lowry of unlabeled membrane and Lipid Gravimetric Analysis)
D. Identity of Proteins Present (from SDS-PAGE)
E. Disposition of the proteins within the membrane (from SDS-PAGE)
IV. Discussion: (1-2 pages, 10 pts)
1) What kinds of surface carbohydrates did you determine are present or not present? What kinds of lipid are present?
2) What is the protein/lipid ratio. Is your lipid/protein ratio different from your expectations? If your result is different, why is it different? How does it compare to other data from your classmates or from other sources you may have come across (site any references)? In general, what factors determine this ratio in cellular membranes and why would the ratio be different in different cell types?
3) On the basis of your data, estimate the molecular weight of each protein. From the protein’s size and the data in your lab manual, text and other sources, try to identify each protein and label it on your gel. Determine which proteins are internal or external and the reasoning that leads you to that conclusion. From information in the text, give a brief description of each protein’s function.
4) Based on your own data and/or that of the rest of the class, construct a schematic diagram of the red cell membrane showing the types of proteins and where they are located (this does not need to be an artistic representation). Stick to information you have derived (be careful not to confuse what information may be inferred from your data and what you may already know from lecture – for instance, did you do an experiment to turn the membranes inside out, reseal them and then label with FITC to prove that some proteins are transmembrane?).
here is some data u may neeed
the tube = 6.6
beaker = 102
tlc paper
14.5
6.4 LE
11.7PE
9.4PC
4.5S
14.2 PE
15.3 PS
14.5LE
=2.26
