The Electrophoresis of Human Hemoglobin
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The Electrophoresis of Human Hemoglobin

Linda H. Austin

Target age or ability group: High school biology students.
Class time required: Two to five forty-five minute periods depending on the extent of pre- and post-lab activities and extensions. Allow one class period for each of the following:

  • practice using digital micropipets or some other gel loading device and pouring the gel
  • loading the hemoglobin samples and running the gel
  • Coomassie blue staining and destaining (can be done overnight)
  • recording and analyzing the gel results and completing the pedigree chart
  • extension activity: transcription and translation of portions of the DNA sequences for normal and sickle cell hemoglobin
Materials and equipment: power supply

horizontal electrophoresis chamber, casting tray, comb

digital micropipet, disposable tips, or some other implement to load the gel

staining dish, disposable gloves

running buffer (1X Tris-Glycine)

1.5% molten agarose

samples containing normal adult hemoglobin, sickle hemoglobin, and a mixture of the two types

Coomassie blue stain

destaining solution

Summary of activity: In this activity, students are presented with a scenario that requires them to electrophorese human hemoglobin samples in order to confirm a diagnosis of sickle cell anemia and/or to determine whether individuals in the scenario are carriers of the sickle cell allele. Students will be asked to analyze the separation of the different types of hemoglobin on the gel. From this analysis, they will be able to determine the alleles/genotypes of individuals in a pedigree chart. As an extension of the activity, students can be asked to transcribe and translate portions of the DNA sequences for the two alleles. Also as an extension, students can be presented with questions about how the Hardy-Weinberg equation applies to the alleles for normal and sickle hemoglobin in human populations.
Prior knowledge, concepts or vocabulary necessary to complete activity: Some understanding of Mendelian genetics is essential. The alleles for human hemoglobin are codominant. In this activity, students are asked to interpret the results on the gel and use the information to construct a pedigree chart. Students need to understand the principles of electrophoresis. Prior hands-on experience with agarose gel electrophoresis is desirable, but not necessary because this is a "load and run" lab; however, students will need to practice using the digital micropipet or whatever implement is used to load the gels. If the DNA sequence extension is selected, students will need to understand the DNA code, transcription, and translation. Some prior knowledge is required if the Hardy-Weinberg extension is selected.

Teacher Instructions

Hemoglobin Sample Preparation

(1) Hemoglobin can be purchased from some chemical supply companies. Most require a letter on school letterhead explaining how the hemoglobin will be used. These blood products are routinely screened for both AIDS and Hepatitis B, but standard precautions should be used: GLOVES AND BLEACH CLEAN-UP. The following instructions are based on the 100 mg size.

(2) Prepare 500 mL total volume of a 1.5M solution of Tris (Sigma #T1503), pH=9.2.

(3) Dissolve each of the hemoglobin samples in 20 mL of the 1.5M Tris, pH=9.2.

(4) PREPARE A 2X Sample Buffer by mixing the following:

    glycerol (#G6279) 8.00 mL
    1.5M Tris, pH=9.2 4.00 mL
    deionized water 28.00 mL
    bromophenol blue (#B8026) 0.01 gm

(5) Add 20 mL of 2X Sample Buffer to the dissolved HgA, mix and dispense into 5 mL aliquots. Place in the refrigerator for short term storage (one month). For storage longer than a month, place in a freezer (can be frozen indefinitely).

(6) Add 20 mL of 2X Sample Buffer to the dissolved HgS, mix and dispense into 5 mL aliquots. Store appropriately. (See Step 5.)

(7) For each group of students, aliquot 20 µL of each sample. Students will load 15 µL. To prepare the sickle trait samples, mix 10 µL of HgA and 10 µL of HgS. Student samples can be stored for one week in a refrigerator.

Tris-Glycine Electrophoresis Buffer

(1) For each station, prepare 500 mL of Tris-glycine electrophoresis buffer by mixing the following:

    Tris base 8.0 gm
    Glycine 3.6 gm
    deionized water 500.0 mL

(2) The electrophoresis buffer can be stored indefinitely. Place in a refrigerator if possible.

Agarose Gels

(1) Prepare 1.5% agarose gels by mixing 1.5 gm of agarose and 100 mL of Tris-glycine buffer.

Each group of students will use approximately 30-50 mL of agarose.

(2) To dissolve the agarose, bring the mixture to a boil in a microwave oven or double boiler.

Coomassie Blue Stain

(1) Mix:

    40.0% methanol
    10.0% acetic acid
    0.5% Coomassie blue
    49.5% distilled water

Destaining Solution

(1) Mix:

    10% methanol
    10% acetic acid
    80% distilled water

Possible scenario: Parents have brought their youngest daughter to the genetics clinic to be tested because their family doctor suspects that her chronic health problems may be caused by sickle cell anemia. The genetics counselor meets with the family and suggests that all members of the family be tested. The family consists of the mother, father, two older sisters, and the youngest daughter. By electrophoresing their hemoglobin samples, it will be possible to confirm the diagnosis of sickle cell anemia and identify carriers of the allele. A control sample containing both types of hemoglobin should be run on the same gel.

References:

Cahill, Holly. "An Interdisciplinary Look at Sickle Cell Anemia." Woodrow Wilson Middle School Biology Institute, 1994.

Capra, Judy. Genetics: A Human Approach, Rev. Ed. University of Colorado Health Sciences Center, 1983.

"The Mystery of the Crooked Cell." CityLab, Boston University School of Medicine, Boston, Massachusetts, 1993.

Offner, Susan. "Dry Lab #2." American Biology Teacher, February 1992.

Pines, Maya. "Turning Back the Biological Clock to Cure Sickle Cell Disease." Blood: Bearer of Life and Death. Howard Hughes Medical Institute, 1994. 18-27.


Lab No.Date Name

The Electrophoresis of Human Hemoglobin Molecules

INTRODUCTION: Two variants of human hemoglobin, normal and sickle cell, are examined in this activity. In homozygous (pure) condition, two alleles for sickle cell hemoglobin result in a serious medical condition known as sickle cell anemia. In the United States, eighty thousand African-Americans suffer from sickle cell anemia. Two and a half million African-Americans (8%) are carriers of the sickle cell allele. In some areas of Africa, 30% of the people are carriers of the sickle cell allele. Such high frequencies of an otherwise harmful allele suggest some type of selection pressure. Population studies have demonstrated a link between carrier status and resistance to malaria.

A single dose of this gene offers some protection against malaria, which is common in Africa. When malaria parasites invade the bloodstream, the red cells that contain defective hemoglobin become sickled and die, trapping the parasites inside them and reducing the infection. As a result, some 30% of the people in certain areas of Africa have the sickle cell trait (carrier status). The trait is also widespread around the Mediterranean and in other areas where malaria used to be a major threat to life (Pines 24).

OBJECTIVES:

Use the technique of agarose gel electrophoresis to separate human hemoglobin molecules according to their electrical charge, size, and/or shape.

Relate the migration of hemoglobin molecules on the gel (phenotype) to the alleles possessed by individuals in the scenario (genotypes).

MATERIALS:

In the space provided below, list the equipment and supplies that you and your partner(s) used to perform this activity.









PROCEDURE:

(1) POURING THE GEL
(a) If your casting tray has gates, use them to close off the ends. If your tray does not have gates, use masking tape to dam both ends. Place the comb in the center slots.
(b) Place the casting try where it will not be disturbed.
(c) Pour the melted agarose gel solution to a depth one third to one half the way up the teeth of the comb.
(d) Do not disturb the gel for 10-15 minutes.
(2) LOADING THE GEL
(a) When set, the gel will have a slightly milky appearance.
(b) Carefully lower the gates or remove the masking tape from both ends of the casting tray.
(c) Immerse the casting tray into the electrophoresis chamber . Add the running buffer to the chamber so that it just floods the surface of the gel.
(d) Remove the comb, taking care not to tear the wells created by the comb.
(e) Use a digital micropipet or other implement to load 15 µL of each hemoglobin sample into separate wells. Avoid the wells on either end whenever possible.
(3) RUNNING THE GEL
(a) Close the cover on the electrophoresis chamber.
(b) Attach the leads to the power supply, red to red and black to black.
(c) Select the appropriate channel on the power supply and adjust the voltage to 150 volts.
(d) Every 10 minutes, turn off the power, remove the cover from the chamber, and observe the progress of the hemoglobin molecules through the gel.
(e) Run the gel for 30-60 minutes at 150 volts.
(4) STAINING THE GEL

NOTE: Time permitting, you may do some or all of the steps in the staining procedure. Your teacher will complete the procedure.
(a) Turn the power supply off.
(b) Detach the leads from the power supply and remove the cover from the electrophoresis chamber.
(c) Remove the casting tray and slide the gel into the staining dish.
(d) USE DISPOSABLE LATEX GLOVES to pour Coomassie blue stain over the gel. NOTE: Coomassie blue is a protein stain; in addition to staining hemoglobin, it will also stain your skin!
(e) Stain the gel for 1 hour.
(f) Replace the stain with destaining solution. For best results, change the destaining solution several times. Destain the gel until the "bands" of hemoglobin are clearly visible on the gel (1-24 hours). High school biology students.
(5)
VIEWING THE GEL
(a) USE DISPOSABLE LATEX GLOVES to place the gel on a sheet of clear plastic. Observe the gel on a lighted surface such as an overhead projector, slide viewer, or white light transilluminator. High school biology students.

RESULTS:

Record the results of electrophoresis on the gel template below. Draw each of the wells that the comb created and label each of the wells into which sample was loaded. Indicate the direction and distance that the hemoglobin molecules migrated from each well.
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ANALYSIS OF RESULTS:

Base your answers to the questions below on your analysis of the gel and on the following information.

The samples contained two types of hemoglobin, normal and sickle cell. Both normal and sickle cell hemoglobin have the same size molecules containing about 600 amino acid subunits. However, there is a single amino acid substitution between the two. In normal adult hemoglobin, the sixth amino acid in the beta chain is a negatively charged subunit. In sickle cell hemoglobin, the sixth amino acid in the beta chain is a nonpolar amino acid having no charge.

(1) From the results shown on your gel, is the overall charge on the two types hemoglobin molecules positive, negative, both, or neither? How do you know?







(2) From an analysis of the results, which of the samples contains only sickle cell hemoglobin? What is your reason for choosing this one?







(3) From an analysis of the results, which of the samples contain(s) a mixture of normal and sickle cell hemoglobin? Explain your reasoning.







(4) Construct a pedigree chart for the members of the family investigated by your group. Show the genotype for each member of the family based on your analysis of the electrophoresis gel. Use the genotype key below:

A = allele for normal adult hemoglobin (normal beta chain)

S = allele for sickle cell hemoglobin








References:

Pines, Maya. "Turning Back the Biological Clock to Cure Sickle Cell Disease." Blood: Bearer of Life and Death. Howard Hughes Medical Institute, 1994. 18-27.

Human Hemoglobin Extension Activity

(1) The DNA sequence below is the beginning of the coding region for the beta chain of normal human hemoglobin. Write the messenger RNA (mRNA) sequence that would be transcribed from this template DNA sequence:

C A C G T G G A C T G A G G A C T C C T C








(2) Circle the sixth codon in both the DNA and mRNA sequences.







(3) Write the amino acid sequence that would be translated from the mRNA sequence.







(4) Circle the sixth amino acid in the polypeptide.







(5) The mutation that produces sickle cell hemoglobin is a single base substitution (point mutation) in the sixth codon. The mutation results in the replacement of a negatively charged amino acid (glutamic acid) with a nonpolar amino acid having no charge (valine). Using the translation table, propose point mutations in the sixth codon that would result in the substitution of valine for glutamic acid.







(6) If the beta chain of human hemoglobin is 146 amino acids in length, calculate the minimum number of nucleotide base pairs needed to code for beta globin.







On to Electrophoretic Analysis:
DNA Fingerprinting and Evolutionary Divergence

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