Wayne State University School of Medicine - Yearbook (Detroit, MI)

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INTRA-ERYTHROCYTIC LOCUS OF HEMOGLOBIN USING FLUORESCENT ANTIBODY TECHNIQUES As part of a continuing project in the Department of Biochem- istry to identify the intra-erythrocytic locus of hemoglobin and the subcellular site of hemoglobin synthesis, new techniques were developed replacing those previously reported CNelson and Orten, 1966 Yearbookj. Realizing that acetone extraction of the heme of hemoglobin, countercurrent separation of the alpha and beta chains and lyophillyzation led to a denatured product unsuitable for use as an antigen. The following procedure was adopted. Human erythrocytes were washed, lysed, and the hemoglobin purified by fractional precipitation with ammonium sulfate. The alpha and beta chains of hemoglobin were separated using pH gradient elution over a carboxymethylcellulose column. The beta chains were then concentrated and injected into the foot pads and subcutaneously into the backs of Dutch rabbits. After a suit- 'able period, the rabbits were sacrificed and the gamma globulin fraction containing the anti-human-beta-chain antibodies was iso- lated. The purity of the preparation was tested by immunoelectro- phoretic techniques and the titer of antibodies established. Finally, human erythrocytes were incubated with the rabbit anti-beta chain antisera, washed, then re-incubated with sheep anti-rabbit globulin-fluorescein conjugated antisera. These cells were then examined under the fluorescent microscope where pre- liminary results indicate that the beta chains Cand therefore the hemoglobinl are located uniformly throughout the mature erythro- cytes. It will be the purpose of a subsequent project to determine the sites of hemoglobin synthesis in the erythrocyte precursors using this method. Roger F. Suchyta james M. Orten, Ph.D.

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l I AGAR-GEL ELECTROPHORETIC TECHNIQUE FOR SEPARATION OF NUCLEOBASES FROM DEOXYRIBONUCLEIC ACID An agar-gel electrophoretic technique for the separation of nucleobases from purified deoxyribonucleic acid CDNAD was developed. This method is an adaptation ofa method previously employed for rihonucleic acid. DNA was extracted from a variety of microorganisms and was analyzed by two of the common techniques, thermal denaturation and buoyant density. Samples of these DNA's were then hydrolyzed with formic acid and the nucleobases spotted in high concentration on the agar gel and subjected to an EMF of 250 volts. This gave separation. The bases were then eluted from the electropherogram and quantitated by their characteristic absorption in ultra-violet light. Such a technique is important in genetic studies, where great emphasis is placed upon the DNA's molar percentage of guanine plus cytosine in determining relationships between organisms. This technique has the advantage of being simple and rapidly performed, while maintaining the accuracy ofthe more complex determinations. Perry Seese L. NI. Weiner, Ph.D. rg. .. 4 YW fs-,-.K f if 551, f ' ass, ' S 'f 2 W E tt . 'a 5 . - K i Q' N ill v Y W .- -. ., 1f'.i , WAV , ii iii: , l h,-TTAEB '- -- - . . W ,,. .... 4: . . 7 if I - E J H y 1 t D .4 .. ' 'i' x ' r 'Tis' ,af Q ig!!! .,i v can fi- , .,,.,.,,,..., my , . I V s....., ., ,EE . .H --ft sky Lg! L Y . ty -rw s - vi ..... .4 - x I Q Q it K A ' A i R' 15 1 Q 1 iv.. 1-, ' J .S .... 1 N, 1 'Su A- erik it ' ' O' i - ' 1e 'ifi - t..2:f i.' H I A

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