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A reduction of __________ in the mouse red blood cell membrane may result in

anemia and spherocytosis.

Insert your name

Insert your email address

Insert your section number and TAs nameDepartment of Biological Sciences

Insert the date

Introduction Type in your answers to the questions below.

1. Spherocytosis is a form of hemolytic anemia wherein red blood cells have a

spherical shape, rather than the normal bi-concave shape. As a result, these redblood cells do not live as long as normal ones and the bone marrow cannot

replace these cells fast enough, leading to an RBC deficiency.

2. Several protein deficiencies have been linked to spherocytosis. These in includespectrin (Peter 1985), ankyrin & band 3 (Eber 1996), and band 3 & spectrin

(Mariani 2008).

3. The objective of this project is to determine, through gel electrophoresis, which

protein deficiency can be correlated to spherocytosis. What is the objective of this

project?

Materials and Methods This section should be written in formal scientific paperformat. See also pg. 52-55 Writing Handbook; Sample Materials and Methods section

posted on Concourse. For final draft, delete all instructions.

RBC membranes isolated.500 microliteres of mouse blood were centrifuged for 5 min at 2,500 x g. The plasma was

separated from the RBC's. 1000 microliters cold wash buffer were added to the RBC's.

The cells were then centrifuged for 5 minutes at 2,500 x g. Cells were then lysed in 15mlcold hemolysis buffer. The lysed cells were centrifuged for 30 minutes at 16,100 x g.

1000 microliters cold hemolysis buffer was added to membranes. The cells were

centrifuged for 15 minutes at 16,100 x g.

Laemmli buffer added to RBC membranes

Hot water added to wellsTubes heated in hot water

gel prepeared

gel electrophoresis run

coomassie stain performedwestern blot performed

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Essential details include: speeds, times, and buffer information. Omit references to

containers, pipettes, routine procedures, and to class or lab. You should also omit

steps that describe decanting, etc. - if it is obvious from your statement that you arecollecting the pellet, you do not need to mention the supernatant. Target length for this

section is one short paragraph.

Summarize the procedure used for SDS-PAGE and Coomassie staining. Essential details

include: sample preparation, gel and buffer information and where appropriate,

manufacturer information. SDS-PAGE is a standard lab procedure so this descriptionshould be brief. Details such as chamber preparation, lane assignments or loading

instructions do not need to be included. For Coomassie staining, you do not need to

repeat the procedure since the instructions come with the stain itself. You should

however cite the name and manufacturer of the stain, and in doing so you have providedthe reader with the necessary protocol information. The entire SDS-PAGE and staining

procedure should not exceed 2-3 sentences.

The sample was prepared with 30 microliters Laemmli buffer. Gel electrophoresis wasperformed using gel from Bio-Rad Laboratories. A coomassie stain was then performed

with coomassie from Bio-Rad Laboratories.

Summarize the procedure used for transfer and Western blot. Essential details include:

type of membrane paper; primary antibodies, secondary antibodies, and developer alongwith manufacturer information. As with SDS-PAGE, a Western blot is a common lab

procedure, so details about the physical set up etc are not required. Again, 2-3 sentences

should be sufficient.

Tranblotting sandwich prepared with .2 micrometers of nitrocelluose. The western blot

was performed using Band 3 (primary) and mouse (secondary) antibodies. NBT/BCIPtablets used to develop blot.

The Materials and Methods should be written in past tense and should not use 1st person.Section subtitles may be used to organize your information.

Results - This section should be written in formal scientific paper format. See also

Handbook pg. 55-59; Sample Results section posted on Concourse. For final draft,

delete all instructions.

Produce your two figures first. Each figure must have a figure caption. A completecaption should contain: (1) the title, (2) a statement about the method used to obtain the

data (1 sentence) and (3) the general trend or idea presented in the figure (1-2 sentence).

Figure 1 This figure should include an annotated picture of your Coomassie stained

SDS-PAGE gel. Lane numbers or other labels should be added to the picture so the

reader knows the identity of samples in each lane. Also identify alpha spectrin, beta

spectrin and Band 3 protein on the gel. Ankyrin is hidden behind the spectrin so it is not

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necessary to identify this protein. (Powerpoint is a relatively easy way to annotate a gel

picture. The edited picture can be copied and pasted into Word.)

Figure 2 This figure should include an annotated picture of your Western blots. You

may use an A, B, C designation to identify the different blots. As with the Coomassiegel, add labels so that the reader knows the identity of each sample.

Figures are also accompanied by a separate text section. The text should describe thegeneral results and trends that are detailed in your figures. Cite the appropriate figure in

parentheses within the text.

The Results should be written in past tense and should not use 1st person. Be concise and

direct - see Handbook pg. 58-59 for recommendations. In your final draft, please have

the text section first followed by Figure 1 and 2.

Discussion - Type in your answers to the questions below.

1. Relate your results back to the objective of your experiment. What do yourresults tell you?

2. Why would reduction of this protein(s) lead to spherocytosis and anemia?

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3. If you were to keep working on this particular mouse strain, what would be your

next experiment? Why? If you dont know the name of a particular technique,you may simply describe the research objective.

The majority of the Discussion should be written in past tense. A limited use of use 1st

person is permitted as described in the Writing Guide.

Submission Instructions

Please use the following file name format: Your last name RBC membrane report (ex.

Lewis RBC membrane report).

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