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 TA’s name
Department 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 red
blood 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 include

spectrin (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 paper
format. 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 15ml
cold 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 wells
Tubes heated in hot water
gel prepeared
gel electrophoresis run
coomassie stain performed
western blot performed
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 are
collecting 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 description
should 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 provided
the 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 was
performed 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 along
with 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/BCIP
tablets used to develop blot.

The Materials and Methods should be written in past tense and should not use 1 person.
st

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 complete
caption 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
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 Coomassie
gel, 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 the
general 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 1 person. Be concise and
st

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 your
results tell you?

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

 3. If you were to keep working on this particular mouse strain, what would be your
next experiment? Why? If you don’t 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 1 st

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).