MAMMALIAN HEMOGLOBINS 305 



although the quantity of A 2 hemoglobin may increase under the influence of other 

 types of anemia. 671 Cepellini, 187 through the use of starch-block electrophoresis, 

 has recently identified a new human-hemoglobin fraction (B 2 ) which he suggests may 

 be the product of an allele of the locus that controls the synthesis of A 2 hemoglobin in 

 humans. 



3. Starch-gel electrophoresis: Resolution on starch gel is greater than that on 

 paper. The adsorption of proteins on starch gels is low, similar to that for starch-block 

 electrophoresis; the gels are easy to prepare and can be preserved for permanent 

 records. Another advantage of the starch gels over paper is that the rate of migration 

 is less dependent on concentration of protein. Furthermore, samples being compared 

 can be run adjacent to one another on the same starch-gel block without appreciable 

 interference by lateral diffusion of the proteins during electrophoresis. The design of 

 the apparatus, preparation of gels, application of material, staining, and other details 

 of the method for starch-gel electrophoresis have been described by Smithies. 1228, 1229 

 The technique is not so simple as paper electrophoresis, but the increased resolution 

 achieved more than compensates for its increased complexity. Prehydrolyzed starch 

 has recently become commercially available, 1227 thus removing a major source of 

 variability. Twelve to 16 g. of starch per 100 ml. of buffer is heated until the opaque 

 mixture becomes transparent and thick. The solution should not be removed at this 

 point but is heated longer and shaken vigorously until it begins to become less viscous. 

 The air bubbles are moved by placing the heated solution under reduced pressure for 

 few seconds using a flask with heavy walls before the liquid starch is poured into plastic 

 trays. A cover is placed on each tray removing excess starch and producing a uniform 

 starch-gel block. Optimal gelling is achieved by placing the preparations in 

 a refrigerator at 4-6° C. overnight. 



Boric acid-NaOH buffers, 0.015-0.04 ionic strength, pH 8.0-8.6, are most common- 

 ly used, but other buffers, such as formate, 0.02 ionic strength pH 1 .9, 918 phosphate, 1228 

 and acetic acid-sodium acetate (unpublished) have been used with hemoglobin. The 

 gelling property of the starch is influenced by the type of buffer and the amount of 

 starch used ; thus, the percentage of starch may need to be changed when a new buffer 

 is tried. 



Several methods have been used to apply samples to the starch-gel preparation. 

 Samples to be inserted may be mixed with starch to make a thin paste that is pipetted 

 into a slot made in the gel, or samples may be applied to filter paper that is placed 

 into a transverse cut made in the gel. The slot method of application is recommended 

 when larger samples are required; however, resolution under such conditions may be 

 somewhat reduced. The quality of paper, when the technique of paper insertion is 

 used, is important; if the paper adsorbs hemoglobin tightly, the bands are spread more. 

 A thin plastic sheet is placed over the gel to reduce evaporation during electrophoresis. 



The electrophoretic patterns are better near the center of the gel ; therefore, the 

 starch-gel slabs are sliced horizontally and the hemoglobin patterns at the inner surfaces 

 are compared. It may not be necessary to stain hemoglobin owing to its red color, 



