Hemoglobin Electrophoresis

Robert G. Hamilton and Prentiss G. Cox

Department of Biological Sciences, Mississippi College, Clinton, MS 39058

We outline a method by which polyacrylamide gel electrophoresis (PAGE) can be used to separate blood proteins and to identify hemoglobin in particular. Blood samples can be subjected to either reducing PAGE or native PAGE. Reducing PAGE will result in the denaturation of the hemoblobin into the alpha and beta globin subunits. Native page will not result in the denaturation of hemoglobin. This is a quick, easy, and effective excercise that provides students with an enhanced understanding of protein electrophoresis, greatly increasing their ability to understand information involving an interpretation of gels.

When developing laboratory activities for undergraduates, we try to use available resources to develop exercises that are as representative as possible of current research and diagnostic protocols (e.g., Hamilton, 1997). We look for exercises that are not technically difficult nor overly expensive. Electrophoresis is widely used as a method for analyzing large organic molecules such as polypeptides and polynucleotides (e.g., Sambrook et al, 1989). As in most college campuses, research laboratories at Mississippi College include electrophoresis equipment. In this article we will describe a method whereby electrophoresis equipment on-hand in most college campuses can be used for a simple, elegant laboratory exercise involving the use of polyacrylamide gel electrophoresis (PAGE).

Red blood cells consist of large amounts of the protein hemoglobin, and as such provide an excellent, readily obtainable supply of a well known protein for a laboratory exercise. We use student's and instructor's own blood. However, any whole blood can be used. Hemoglobin is very easy to extract from whole blood and analyze using PAGE. Specialized equipment needed for the methods to be described are an electrophoresis chamber and a power supply. We use equipment specialized for PAGE.

MATERIALS AND METHODS

To extract hemoglobin, an auto-lancet and tip are used to pierce the skin of a fingertip and a micropipetter with a sterile tip is used to collect 10 µl of blood. Auto lancets, tips, and the alcohol swabs used when drawing such blood samples are available at any pharmacy. The 10 µl of blood is then mixed with 40 µl of an isotonic solution (0.100 M NaCl) in a 500 µl microcentrifuge tube and centrifuged for 2 minutes at 6500 RPM (approximately) to pellet the red blood cells. The blood serum is the supernatant, which is transferred to another 500 µl microcentrifuge tube and saved. The pellet of red blood cells is resuspended in 40 µl of a hypotonic solution (0.065 M KCl) and vortexed vigorously to lyse all cells. The lysed red blood cell solution is then centrifuged at 13000 RPM (approximately) for 5 minutes to remove any undissolved materials (such as membranes). There are now two samples, a serum sample and a red blood cell sample.

The samples are now ready for PAGE. Two types of PAGE can be used, native PAGE or denaturing PAGE. If native PAGE is used, the intact hemoglobin tetramers will be observed. Each hemoglobin tetramer consists of 2 alpha and 2 beta globin subunits. Each alpha globin subunit consists of 141 amino acids, and has a molecular weight of 15,126 Daltons. Each beta globin subunit consists of 146 amino acids, and has a molecular weight of 15,867 Daltons. If denaturing PAGE is used, the alpha and beta globin subunits will be observed. Denaturing PAGE gels contain sodium dodecyl sulfate (SDS), a detergent, and thus is commonly described as SDS-PAGE. Prior to loading samples onto gels, samples (either serum and/or red blood cell) must be mixed with sample buffer. If SDS-PAGE is to be used, solutions must be mixed 2:1 with Laemmli sample buffer, and, if native PAGE is to be used, the blood solution must be mixed 2:1 with native sample buffer prior to loading them onto a gel.

We use an electrophoresis chamber that allows for 8 cm gels. The gels that we use are pre-made 4­20% linear gradient gels, with no SDS. For denaturing PAGE, SDS need only be present in the running buffer and the sample buffer. We purchase sample buffer and running buffer pre-made. Running buffer (either native PAGE or SDS-PAGE) is a 10X concentration, and must be diluted prior to use. The pre-made gels snap into place in the electrophoresis chamber, which is then filled with running buffer.

Controls must be loaded onto gels along with samples. With native PAGE gels, we have used AFSC controls, containing hemoglobins A, F, S, and C. Hemoglobin A is normal adult hemoglobin, hemoglobin F is fetal hemoglobin, hemoglobin S is the sickle cell associated hemoglobin and hemoglobin C is hemoglobin with the mutant -6-Glu-Lys beta globin subunits. We use molecular weight markers (either small or broad size range) when running SDS-PAGE gels. Thus with native PAGE gels we load two lanes with markers and six lanes with samples on each gel, and with SDS-PAGE gels we load one lane with molecular weight markers and seven lanes with samples on each gel. We usually include at least one lane with a serum sample for comparison to the red blood cell sample. Molecular weight markers and AFSC controls must be diluted at least 2:1 with sample buffer prior to loading. We load 5 µl of markers and 10 µl of samples. Samples are electrophoresed at 200 volts for 30 minutes.

Following electrophoresis, gels are stained using Coomassie blue R-250, destained and viewed. Staining and destaining are completed using standard protocols which are described in documentation supplied with the electrophoresis equipment and the pre-made gels. These complete protocols are also fully described in Sambrook et al. (1989).

In SDS-PAGE gels, distance migrated will be a function of the size of the polypeptide. In native PAGE gels, both the size and the charge of the polypeptide will affect migration. Thus the differences in size between the alpha and beta globins cannot be resolved using the methods described in this paper, and the alpha and beta globin subunits appear as one band in SDS-PAGE gels. In native PAGE hemoglobins A and S will migrate differently due to differences in charge, despite the fact they are the same size. Ideally an exercise would involve both types of PAGE. If serum samples are loaded, the principle protein component in serum is albumin, which can be observed in both SDS and native PAGE gels.

When SDS-PAGE is used, molecular weights of proteins can be estimated graphically on semi-log graph paper. Distance migrated is plotted on the linear scale (the x-axis) and molecular weight is plotted on the log scale (the y-axis). Points are plotted for each of the standards (molecular weight markers), and a standard curve is drawn. The distance migrated is then used to estimate the molecular weight of unknown proteins from the standard curve.

We have saved data from the gels by placing the gels between acetate sheets and making a photocopy of the gels. This works very well when the banding is sharp, however the photocopier does not make good images of smeared bands.

If you wish to save the gels they will have to be dried. We dry the gels by placing them between two sheets of cellophane and placing them in a drying chamber. Cellophane sheets must be soaked in water, with one sheet (the bottom sheet) placed on a flat surface. The gels are then placed on the bottom sheet. The bottom sheet and the gels must be covered with a layer of water prior to covering them with the top sheet. All air bubbles must then be removed and the gels dried for 60 minutes. If a drying chamber is not available, the gels could be dried with a hand-held hair dryer. The cellophane/gel assembly cannot be removed from the flat surface until the gels are completely dried. Some means of pressing or clamping the edges of the cellophane sheets together is required.

We use the mini-Protean II system supplied by BIO-RAD (200 Alfred Nobel Drive, Hercules, CA 94547), which includes a 300 volt power supply for $820.00 (all prices quoted are from the BIO-RAD 1997 U.S. catalog). A system designed for use with pre-made gels, including the power supply, is available for $690.00. If you already have an appropriate power supply, the electrophoresis cells are $490.00 for the regular cell and $350.00 for the cell specialized for pre-made gels. The pre-made gels are more difficult to align in the electrophoresis chamber of the regular system, and the upper buffer tank will leak if the gels are not properly aligned. The gel drying system we use costs $695.00.

All buffers and gels can be purchased pre-made. We have found that the convenience of pre-made gels and buffers greatly outweighs their costs. If you have no other use for the components of the gels and buffers, it might actually be cheaper to purchase pre-made buffers and gels than it would be to purchase the components and make them yourself (given a limited shelf life for some components and the probability of contamination of reagents). We have used 1-year old gels and 1-year old buffer, obtaining good results, despite the rapid expiration of gels (as suggested by expiration dates). The problem with expired gels is degradation of the gel at the bottom of the gel. We have not noticed an effect at the middle of the gel, where the bands are located.

The running buffer for the SDS-PAGE is tris-glycine-SDS, and the running buffer for native PAGE is tris-glycine. One liter of 10X running buffer solution costs $20.00, and you will need about 400 ml of 1X buffer for each pair of gels. The sample buffer used for SDS-PAGE is the Laemmli sample buffer. The sample buffer used for native PAGE is the native sample buffer. 30 ml bottles of sample buffers cost $12.00 each. We have used the 4­20% linear gradient gels, which are sold in packages of 10 gels for $82.00. The gel packages also contain instructions for preparing all buffers, staining, and destaining solutions. We use the broad range molecular weight standards, which cost $82.00 per 200 µl (We use 5 µl per lane, and generally use three lanes per gel). We have obtained hemoglobin standards from Helena Laboratories (Beaumont, Texas), however, they are also available from Beckman Instruments, Inc. Diagnostic Systems Group, (Brea, CA 92621-6209). The hemoglobin standards degrade rapidly, resulting in smeared bands on the gel.

The staining solution requires Coomassie blue R-250. A kit which includes pre-made stain and destain can be purchased for $90.00. However, the staining and destaining solutions are easy to prepare and thus we make our own. The staining and destaining solutions require methanol, glacial acetic acid and water. A 10 g bottle of Coomassie blue R-250 costs $16.00. The staining solution can be reused, and thus a 10 g bottle of Coomassie blue R-250 will last a long time. Reagent costs total about $250.00 if all reagents are purchased pre-made.

The specialized equipment and reagents that we have on hand for this experiment are supplied by BIO-RAD as they offer a complete line of equipment and reagents appropriate for PAGE of proteins. Pharmacia (800 Centennial Avenue, P.O. Box 1327, Piscataway, NJ 08855-1327) also offers a similar line of equipment and reagents, which are comparably priced and of at least equal quality.

RESULTS

We used 1 year old gels and AFSC controls stored in a refrigerator, and 3 year old molecular weight markers stored at -10⁰C to analyze human blood from a student in our molecular biology course (Figs. 1, 2). The alpha and beta globins typically form a broad smear at the bottom of the SDS-PAGE gel and the albumin is clearly resolvable between the 45 kD and the 66 kD markers (Fig. 1). Although smeared, the AFSC controls were clearly visible in the native PAGE gel (Fig. 2 ), and both the albumin from the serum and the hemoglobin A from the red blood cells of the blood sampled are easily recognizable.

DISCUSSION

This exercise cannot fail to yield data. The major problem we have had is smearing of bands. Yet even when bands are smeared, it is possible to identify the position of the bands in the control and experimental lanes. The exercise is very easy to perform, and students have always enjoyed it greatly.

There is great concern over the use of body fluids in exercises such as this. However, in this exercise students handle their own blood, and there is no direct contact of the body fluids of one individual with those of another. All materials that come into contact with blood are autoclaved prior to disposal. We do not require students to provide samples during laboratory sessions and still have more than enough volunteers to fully load the 12­14 wells that we have had available for each session. Any individual or institution would have to use their own expertise and judgement when using body fluids such as blood for such experiments.

This exercise has the potential to reveal an individual heterozygous for one of the globin genes, particularly hemoglobin S. We have never had such an event occur, however if it did we would advise any such individual to be tested at an appropriate medical facility.

PAGE of blood as we describe is an elegant and powerful demonstration of the actual methods used to analyze proteins in diagnostic and research laboratories. The use of equipment and reagents we describe requires very little preparation time, and very little technical skill among students. These protocols should be easy to include in courses at most colleges and universities as the equipment is standard in laboratories where investigations involve an analysis of proteins.

We have used this exercise as a demonstration in our sophomore genetics course and as a laboratory exercise in our upper level molecular biology laboratory, human genetics laboratory and our summer workshop in genetics for high school teachers. The experience of a realistic protein electrophoresis exercise greatly increases student's understanding of electrophoresis in general, as well as their ability to understand information (in textbooks, for example) involving interpretations of gels.

LITERATURE CITED

Hamilton, R.G. 1997. DNA sequencing in undergraduate laboratory courses. Journal of College Science Teaching 26:351­354.

Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY.