Name: ______________________________
Biol1A Lab: Genetic Variations in Hemoglobin
Introduction
Access the “Start Here” page for this lab on Canvas. Read and take notes on
the following (posted on Canvas):
1. case study “Sickle Cell Anemia: A Fictional Reconstruction”
2. background information pages “Sickle Cell Disease (SCD)” and
“Inheritance of SCD”
3. genetics vocabulary (gene/allele; genotype/phenotype;
homozygous/heterozygous; dominant/recessive; autosomal/sex-linked)
Materials
Needed per group of 4:
• 50mL graduated cylinder
• electrophoresis running buffer (pH 9.2)
• 125mL flask, sponge cap
• agarose, weigh paper, spatula, balance
• horizontal gel electrophoresis unit, power supply
• 6-well comb
• 3 empty microfuge tubes, rack
• hemoglobin samples (instructor will aliquot to students)
• p20 micropipettor, tips
• microscope slides: normal blood, sickle cell blood (may be set up as
demo)
Methods: prepare, load, and run gel
1. Following your instructor’s demonstration, set up the gel casting tray of the
electrophoresis unit. Place the 6-well sample comb into the tray.
2. Weigh 360mg (0.36g) of agarose. Add to 30mL electrophoresis buffer (pH
9.2). Microwave on high, 20 seconds at a time, until completely melted.
Swirl as needed.
3. Pour the melted agarose into the gel casting tray.
4. Let agarose solidify at room temperature. When ready, the agarose will
appear cloudy.
5. Loosen and push down the casting tray “gates” and place the casting tray
into the gel electrophoresis box.
6. Add approximately 300mL of electrophoresis running buffer (pH 9.2) to the
electrophoresis box. Be sure that there is enough buffer covering the gel.
7. Carefully remove the comb. Ensure that the wells are filled with buffer when
the comb is removed. Return the comb to the front desk ~ do not discard.
8. Label three empty microfuge tubes: “normal”, “trait”, and “sickle.” Get 20µL
aliquots of each sample from your instructor.
9. Using a new pipette tip each time, load 3 separate wells with 20µL of
patient sample (each sample in its own well). There are 6 sample wells and
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Name: ______________________________
only 3 samples, so it is best to use the center wells of the gel. Record which
sample went into each well.
10. Once the samples are loaded, place the lid onto the electrophoresis box
and attach the leads to the power supply (red to red, black to black). Be
sure that the power supply is turned off before doing this step!
11. When all cables are connected, turn the power supply on and set to 100150 volts (current should read 50-100 milliamps).
12. Keep an eye on the blue dye. This will “tell” you when to shut of the
power supply. The dye runs faster than the hemoglobin proteins. Turn off
the power supply when the dye front has reached the bottom centimeter of
the gel. This may take approximately 1 hour.
13. Use this time to observe the prepared microscope slides (normal vs.
sickle cell blood) and answer the post-lab questions, as possible.
14. When finished with the electrophoresis step, turn off the power
supply.
15. Remove the cables and carefully lift the casting tray with gel out of the
electrophoresis box.
16. Slide gel onto a light box and observe the bands of hemoglobin. Sketch
the bands in the results section.
Clean up – did you:
_____ put used pipet tips, weigh paper/weigh boats, microfuge tubes into
trash?
_____ put your gel into the trash (it’s ok, not hazardous)?
_____ rinse dirty glassware and place in appropriate glassware containers?
_____ leave buffer in electrophoresis chambers for the next lab sections to
use; if you are the last lab section, pour buffer back into “used buffer”
container?
_____ rinse gel casting tray and leave to air dry?
_____ wipe down lab bench with bench wash solution?
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Genetic Variations in Hemoglobin
Results and post-lab Questions
1. While your gel is running, answer questions a-e using the information and
data presented in the case study.
a. How many different hemoglobin proteins do you see on the gel?
b. What could cause different rates of migration of the hemoglobin proteins
toward the positive end of the gel?
c. Since all hemoglobin proteins are roughly the same size, what is the
difference between the hemoglobin proteins you see on the gel?
d. If samples 48WC03 and 48WC15 came from patients diagnosed with
SCD, how (specifically) do their hemoglobin proteins differ from the
others?
e. What is a possible explanation for sample 48WC12?
2. Sketch your gel electrophoresis results. Label the sample wells with
“Normal”, “Trait” or “Anemia”. Label each band with HbA or HbS.
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3. According to your results, describe the difference in electric charge between
the sickle cell hemoglobin (HbS) protein and the normal hemoglobin (HbA)
protein. Hint: look at your answer to question 1d.
4. Sickle cell hemoglobin is different from normal hemoglobin only by a single
amino acid change. Review the structures of amino acids (use figure 5.14 in
your text) and give the name of four amino acids which could have replaced
the normal amino acid to give the electrophoretic behavior you saw with
sickle cell hemoglobin.
5. Draw the structure of valine, with its appropriate charges, at pH 2.0
and pH 9.2. Use figure 5.14 in your text to help you ~ but don’t just copy
the figure, the charges shown in the figure are the prevailing charges at
pH 7.2 (cytoplasmic pH).
pH 2.0
pH 9.2
6. Draw the structure of glutamic acid, with its appropriate charges, at pH 2.0
and pH 9.2. Use figure 5.14 in your text to help you ~ but don’t just copy
the figure, the charges shown in the figure are the prevailing charges at
pH 7.2 (cytoplasmic pH).
pH 2.0
pH 9.2
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7. There are other abnormal hemoglobin protein molecules found in the human
population. Can all abnormal hemoglobin molecules be diagnosed by
electrophoresis? Explain your answer.
8. Calculate the net charge on this protein in a solution at pH 2.0. Don’t forget
to add in the terminal amino and carboxyl groups at the ends of the protein!
Use figure 5.14 in your text to help you.
Lys-Ala-Gly-Asp-Asn-Leu-Cys-His-Thr-Glu-Met-Gln-Arg-Tyr-Ile
Net charge = _____
9. Calculate the net charge on this protein in a solution at pH 10. Don’t forget
to add in the terminal amino and carboxyl groups at the ends of the protein!
Use figure 5.14 in your text to help you.
Met—Arg—Gly—Glu—Trp—Phe—Glu—Lys—His—Cys—Met—Ala—Ala
Net charge = _____
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10. Sketches of normal and sickle cell anemia human blood smears– draw
the field of view for each.
Normal Human Blood Smear
Total Magnification = _____
Sickle Cell Anemia Human Blood Smear
Total Magnification = _____
11. What are the chances that offspring of a normal parent and a parent with
sickle cell trait will have sickle cell trait? Use a Punnett Square to answer
this question.
12.
Fill out this table:
Hb b gene
genotype
Allelic
relationship
HbAHbA
HbAHbS
HbSHbS
_____ zygous
_____ zygous
_____ zygous
Molecular
phenotype
(Hb protein)
Cellular
phenotype
(RBC shape)
Organismal
phenotype
(disease
state)
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Name: ______________________________
Figure 5.14 from your text:
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