ANNALS O F CL IN ICAL AND LABORATORY SCIENCE, Vol. 6, No. 2Copyright © 1976, Institute for Clinical Science

A n a ly sis o f H um an Serum P ro te in s by M olecular W eight D ep en d en t Acrylamide G el Electrophoresis

W . C. G R IF F IT H S , P h .D ., H . A. BOGAARS, M .D .,C. F. S H E E H A N , B.S. A N D I. D IA M O N D , M .D .

Department o f Laboratory Medicine, Roger Williams General Hospital, Division o f Biological and Medical Sciences, Brown University,

Providence, RI 02908

ABSTRACTT he te c h n iq u e o f acry lam id e gel e lec tro p h o resis o f sod ium do decy l su l­

fate tre a te d p ro te in m ix tures has b e e n ap p lie d to th e analysis o f hum an serum p ro te in s in th e 70 ,000 to 250,000 m o lecu la r w e ig h t range. After s ta in ­ing, th e ban ds are w e ll d e fin ed , th e m o lecu lar w e ig h t is d e fin ed and, h en ce , th e id en tity o f each can b e e s tim ated from th e m igration d istance . In am biguous cases, th e id en tifica tio n o f a b an d is con firm ed by an in d e p e n ­d e n t m ethod . T h e p ro ced u re is p a rticu la rly v a lu ab le in th e stu dy o f th e gam m opath ies. T h e im m un og lob u lin s m igrate as ex p ec ted , in con trast to th e prob lem s often e n c o u n te re d in th e O m ste in -D av is gel m eth od . F u rth e r­m ore, lig h t chains m ig rate m ore rapid ly , are w ell sep a ra ted from o th e r serum p ro te in s and , th erefo re , a re read ily d e tec ted . P arap ro te in s from th re e p a ­tien ts w ith d o c u m e n te d gam m op ath ies h av e b e e n s tu d ie d an d ch a rac ­te r iz ed u sing th is m ethod .

P o ly a c ry la m id e g e l e le c tro p h o re s is , w h ich com bines th e reso lv in g effects o f o rd inary zone e lec tro p h o resi s w ith th a t o f gel filtra tion , has long b e e n reco g n ized as a po w erfu l separa ting tool in th e stu dy o f com plex p ro te in m ix tu res .4-10 E x cep tin g for s tu d y o f som e isoenzym es in serum , th e p r o c e d u r e h a s n o t b e e n w id e ly u tiliz ed in clin ica l laborato ry p ro b lem s .2,7 A m ajor reason for th is lack o f u tiliza tion d eriv es d irec tly from th e s tren g th o f th e tech n iq u e . W hen s ta in ed w ith a gen e ra l p ro te in dye, serum gives a p a tte rn o f such c o m p le x ity th a t in d iv id u a l b a n d s a re often d ifficu lt to id en tify w ith any ce r­ta in ty . F u rth e rm o re , a g ro u p o f se ru m

p ro te in s o f g reat in te res t, im m un og lob u­lins, G, M an d A, have b e e n re p o rte d to e n te r th e separa tion gels o f th e trad itio n a l a c ry la m id e sy stem in lo w a n d e r ra tic y ie ld . R a th e r , th e s e p r o te in s r e m a in largely above th e separa ting gel, or in th e “ stack ing g e l” w h en one is u se d .6

I t is b e lie v e d by us th a t app lica tio n o f th e s im p le te c h n iq u e o f p r e t r e a t i n g s e ru m p r o te in s a m p le s w i th so d iu m d o d ecy l su lfa te (SD S) m ig h t e lim in a te som e o f th e p rob lem s. T he e lec tro p h o ­re tic m ob ility o f SDS trea ted p ro te in s is m o le c u la r w e ig h t d e p e n d e n t .5'12 T h is m o lecu lar w eig h t d e p e n d e n c e has b e e n sh o w n to b e d u e to a sp ec ific ch a rg e

177

1 7 8 G R IFFIT H S, E T AL.

eq u a liza tio n effect, ow in g to th e b in d in g of th e sam e p e rcen tag e o f SDS to p ro te in , w eig h t to w eigh t, regard less o f th e n a tu re o f th e p ro te in .5,11 T he com plexes assum e a rod-like shap e , an d th e effect o f th e in ­trin sic charge o f th e p ro te in is co n s id e red in sign ifican t w ith re sp ec t to th e an ion ic charac ter o f th e S D S .11 T h is p h en o m e n o n does no t app ly exac tly to all p ro te in s , b u t th e m axim um errors ex p ec ted o f m o lecu ­lar w e ig h t estim atio n in th ose cases are on ly in th e o rder o f 10 p e rc e n t .13 T h u s , in th e e le c tro p h o re s is o f e v e n a com p lex p ro te in m ix ture such as serum , a g iven b an d can b e ten ta tiv e ly id en tified b y its d is tance o f m igration and , h en ce , its cal­cu la ted m o lecu la r w eigh t.

T h e e lec tro p h o resis o f h u m an seru m p r o te in s in S D S -c o n ta in in g p o ly a c ­ry lam id e gels has b e e n ap p lie d p a rticu ­larly to th e stu dy o f th re e p a tien ts — one w ith m u ltip le m yelom a o f an IgG type, a n o th e r o n e w ith m ix e d c ry o ­g lobu linem ia , an d th e th ird w ith chron ic liver d isease w ith p ro d u c tio n o f a m ono­clo nal IgG . T h e first tw o o f th e se patien ts p ro d u ced a c ryog lobu lin ; in th e se cases the p arap ro te in w as p u rified a n d s tu d ied .

M ateria ls an d M ethodsG e l e le c tro p h o re s is w as c a r r ie d o u t

us in g a w ate r coo led Bio-Rad M odel 150 G el E lec tro p h o resis C e ll * in con ju nc tion w ith a B io-Rad m od el 400 or a H ea th k it m o d e l IP -32 p o w er su p p ly .! All e le c ­trop ho resis w as p erfo rm ed at 30 volts and 50 m illiam ps w ith 12 gel colum ns.

S D S -co n ta in in g p o ly a c ry la m id e ge ls w ere p re p a re d in ac id -w ashed 135 m m x 5 m m (I.D .) glass tu bes. T h e p ro ced u re fo llow ed w as th a t o f D u n k e r & R u eck e rt5 for a final to ta l g e l con cen tra tio n o f 5 p e r­c e n t . T h e r e a g e n ts u s e d w e re th o s e su p p lie d in k it form for th is p u rp ose .* T h e gels w ere g en era lly 10 cm in len g th .

* Bio-Rod Laboratories, R ichm ond, CA 94804.f H eath Co., Benton H arbor, MI 49022.

Sodium do decy l su lfate w as p u rch ased com m ercially .*

O n e p e rc e n t io d o a c e ta m id e so lu tio n w as p re p a re d from reagen t. J

U rea (8 M) w as p re p a re d from ana ly ti­cal reag en t g rade urea. §

P h o sp h a te bu ffer (0.1M ) p H 7.2, 0.4 M p h o sp h a te buffer, p H 7.2, 20 p e rc e n t sul- fo s a l ic y l ic a c id a n d 12 .5 p e r c e n t trich lo roacetic acid w e re p re p a re d from th e b e s t ava ilab le q u a lity chem icals. §

T he p ro te in sta in was p re p a re d b y d is­so lv ing 25 m g o f C oom assie b r il la n t b lu e R -250* in 10 0 m l o f 1 2 .5 p e r c e n t tr ic h lo ro a c e tic ac id . P ro te in s tan d a rd s w ere hu m an a lb u m in (m o lecu lar w e ig h t 69,000), IgG (m olecu lar w e ig h t 160,000) an d h o rse -h ea rt cy toch rom e C (m olecu lar w e ig h t 12,500)." T h ese w ere p u re en o u g h to show on ly one b an d for each o f th e s tan d a rd s , p lu s lig h t b a n d s for th e ex­p e c ted d im er, tr im er an d q u a tr im e r o f al­bu m in .

D ialysis w as ca rried o u t in ce llu lo se tu b in g .f

P h o sp h a te b u ffe re d sa lin e is 0.1 M p h o sp h a te buffer, pH 7.2, in 0.9 p e rc e n t NaCl.

A m m o n iu m s u lfa te , a n a ly t ic a l r e a ­gen t, was p u rch ased com m ercially . §

O u ch te rlo n y im m unod iffusion w as car­r ied o u t in gels p rep a red from 1 p e rcen t S pecia l A gar-N oble** co n ta in in g 0.1 p e r­c e n t sod ium az id e .f

Protein electrophoresis was perform ed8 u sin g a B eckm an M icro zo rie .ff

I m m u n o e le c t r o p h o r e s is w as p e r ­fo rm e d a c c o rd in g to th e m e th o d o f C aw ley .3

Q u an titive rad ia l im m un od iffu sion wascarried o u t u sing th e K allestad en d -p o in tp roced ure . $$

$ Sigm a C hem ical, St. L ouis, MO 63178.§ M allinckrodt C hem ical W orks, St. Louis, MO

63160.11 Schw artz/M ann, O rangeburg , NY 10932. f U nion C arb ide Corp., Chicago, IL 60638.

** Difco Laboratories, D etro it, M I 63178. t f Beckm an Instrum ents, Fu llerton , CA 92634.Î Î K allestad Laboratories, Chaska, M N 55318.

ANALYSIS O F HUMAN SERUM 179

S am ple P rep ara tio nS am p les w e re p re p a re d from h u m an

se ru m o r from p u re p ro te in so lu tio n s . E ach sam ple co n ta in ed 0.5 m l 8 M urea, 0 .2 m l o f serum or p ro te in so lu tion co n ­ta in in g 0.2 m g o f each com ponen t, 0.1 m l o f 1 p e rc e n t io do acetam id e so lu tion , 10 m g o f sod iu m do decy l su lfate an d w ate r to b rin g th e to ta l vo lum e to 1.0 ml. Brom - p h en o l b lu e w as ad d ed to one o f each se t o f sam ples as a track ing dye.

T h e u rea, at a final co n cen tia tio n o f 4M , serves tw o pu rp oses. I t is a pow erfu l d e n a tu r in g a g e n t at th is co n c e n tra tio n an d d isru p ts th e secondary , te rtia ry and q u a te rn a ry p ro te in s tru c tu re . F u r th e r ­m ore, i t increases th e d en sity o f th e sam ­p le so lu tion an d facilita tes rep ro d u c ib le a p p lica tio n o f th e sam ple to the top o f th e gel co lu m n by in h ib itin g d iffusion o f th e sam p le in to th e to p b u ffe r lay er. T h e io d o a c e ta m id e s tab iliz e s th e free sulf- hy d ry l g roups o f th e p ro te in b y form ing th e S-carboxym ethyl derivative.

T h e sam p les w e re th e n p la c e d in a w ate r b a th a t 45° for 45 m inu tes. Before ap p ly in g th e sam ple to th e gel, th e tubes w ere p laced in the electrophoresis appa­ratus, and the space above th e gels filled w ith 0.1 M p h o sp h a te bu ffer con ta in in g 0 .1 p e r c e n t S D S . T h e s a m p le s , v o l­u m e 10 i l l , w ere th en lay e red u n d e r th e bu ffe r on th e top o f th e ir resp ec tiv e gels. T h e d en s ity o f th e so lu tion , w h ich is 4M in u rea, re ta rd s d iffusion. T h e top re se r­voir o f th e app ara tu s is th en filled w ith 0.1M p h o sp h a te bu ffe r, an d th e w ate r ja c k e t c o n n e c te d to a co ld w a te r tap . E lec tro p h o res is is ca rried ou t u n til th e m ark er dye has m ig ra ted approxim ately 75 p e rc e n t o f th e len g th o f th e gel.

T h e gels w ere th en rem oved from th e ir tu b es in th e u su a l w ay ,4 fixed overn igh t in 20 p e rc e n t su lfosalicy lic acid, dy ed for a t le a s t e ig h t h o u rs in th e C o o m asie b lu e-trich lo ro ace tic acid so lu tion an d th e b ack g ro u n d c lea red by a llow ing th em to s tan d in 12.5 p e rc e n t trich lo roacetic acid.

T h e gels w ere ph o to g rap h ed on a ligh t box an d scan n ed w ith the D en sico rd d e n ­sitom eter* to p ro d u ce a p e rm an en t rec ­o rd . M e a su re m e n ts w e re m ad e o f th e d is tan ce o f m ig ration o f each b a n d from th e orig in . E ach o f th e se d istances was th en d iv id ed b y th e a lb um in d is tan ce to p ro v id e a R f value.P rep ara tio n o f U rin e Sam ple

To ob tain a sam ple su itab le for e le c ­t ro p h o re s is , u r in e is c o n c e n tra te d by lyop hy lliz ing a su itab le a liquo t, d isso lv ­ing th e re sid u e in a m in im um o f p h o s­p h a te b u ffe red saline, d ia ly z ing against p h o sp h a te b u ffe red sa line a n d c o n cen ­tra ting . All con cen tra tin g was do n e w ith p o lyv iny lpyrro lidone , fP rep a ra tio n o f C ryo prec ip ita te

To 2 m l o f serum w ere ad d ed 2 m l o f co ld p h o sp h a te b u ffe red saline. T his was allow ed to stand at 4° for at least tw o hours an d w as th e n cen trifu g ed at 4° for 15 m in . T h e su p e rn a tan t was d iscard ed , an d th e p rec ip ita te w ash ed w ith p h o sp h a te b u f­fe red saline. T h e p u rity o f th e iso la ted p r o te in w as c h e c k e d b y O u c h te r lo n y d o u b le diffusion.R ecovery o f P ro te in A fter E lec tro p h o res is

E lu tio n o f p ro te in sam ples from SDS- p o ly acry lam id e gels w as acco m p lish ed by cu ttin g ou t th e app ro p ria te sec tio n o f th e gel (as id en tified by com parison w ith a p a ra lle l s ta in ed gel), su sp e n d in g th e sec tion in p h o sp h a te b u ffe red sa line an d d ia ly z ing th e sec tion o v ern ig h t to allow th e sam ple to d iffuse from th e ge l and th e SD S to d iffu se from th e sam p le . T h e d ia ly zed sam ple was first co n cen tra ted to ab o u t 1 m l against p o ly v in y lpy rro lidon e a n d w as th e n b ro u g h t to a v o lu m e o f

* Photovolt Corp., N ew York, NY 10010 f G e n e ra l A n ilin e an d F ilm Co., N ew York,

NY 10020

1 8 0 GR IFFITHS, ET AL.

about 30 jixl w ith an A m icon A25 dialysis concen tra to r.! T he e lu te d p ro te in sam ­ples w ere u sed to check for ban d id en tifi­cation, by O u ch terlony do ub le diffusion, and for recovery by radial im m unodiffu­sion.C alcu lation of M olecu lar W eigh t

A sem i-logarithm ic p lo t was m ade of the R f value (relative to album in) o f the th ree standard p ro te ins vs. th e ir m olecu­lar w eights. F rom th is graph, th e m olecu­lar w eigh t o f p ro te in s g iving bands in the o ther gels was estim ated .R esults and D iscussion

T he m igration rates of the horse heart cytochrom e C (M.W. 12,500) and hum an IgG (M.W. 160,000) standards re la tive to th e h u m a n se ru m a lb u m in s ta n d a rd (M. W. 69,000) w ere qu ite rep rodu c ib le w ith m ean values o f 1.69 and 0.47, re ­

| A m ico n C o rp ., L e x in g to n , M A 02173.

spective ly . T h e stan dard dev ia tions on th e s e m e a s u re m e n ts w e re 0 .1 1 2 a n d 0.028 (N = 10) respectively , rep resen tin g coeffic ien ts o f varia tion o f 6.8 p e rc e n t and 6.0 percent.

R ecovery o f a lb um in and IgG from th e g e ls ra n g e d from 50 to 70 p e rc e n t as show n by rad ial im m unodiffusion. This recovery was ach iev ed by sim ply rem ov­ing th e sam ple from th e dialysis rack after the first concen tra ting step , and w ith ou t special w ash ing o f the m em brane.

In figu re 1 are show n th e resu lts o f SD S-polyacrylam ide gel e lectrophoresis o f th e se ru m o f five h e a lth y h u m an s. U nder the d escrib ed experim en ta l con d i­t io n s , th e n u m b e r o f d is t in g u is h a b le ban d s in norm al specim ens v a ried b e ­tw een 10 and 14 d ep en d in g on th e in d i­vidual. In tab le I is a lis tin g o f th e ten ban d s w h ich ap p ea r d is tin c tly in m ost sera . N o te th a t th e s e d is t in g u is h a b le b an d s are all in th e m o lecu la r w e ig h t range o f 70,000 to 300,000. In c lu d ed is an

F ig u re 1. S ta n d a rd (left) a n d re su lts o f e le c tro p h o re s is o f five n o rm al su b je c ts . S ta n d a rd c o n ta in s h u m a n IgG , h u m a n se ru m a lb u m in , a n d h o rse h e a r t c y to c h ro m e C. F ig u re 2. S ta n d a rd (left), d u p lic a te e le c - tro p h o re to g ra m s o f th e se ru m o f W . P ., a n d e le c tro p h o re to g ra m o f th e c ry o p re c ip ita te from th e se ru m . T h e s e sa m p le s w e re a n a ly z e d d u r in g d if fe re n t e x p e r im e n ts a n d w e re ru n fo r d i f fe re n t tim e s .

ANALYSIS O F HUMAN SERUM 181

id en tifica tio n o f ce rta in o f th e ban ds. T he in itia l ten a tiv e id en tifica tio n w as m ade on th e b a s is o f c a lc u la te d m o le c u la r w e ig h t an d e s tim a ted q u an tity com pared to kn ow n co n cen tra tio n ranges in norm al sera. P ro te in s o f lo w er m o lecu lar w e ig h t than th is g roup m ig ra te too far to be d e ­te rm in ed by th is m ethod . L ik ew ise , th e h eav ie r p ro te in s b u n ch at th e to p o f th e gel an d are no t reso lv ed . T heo re tica lly , th e lig h te r an d h ea v ie r p ro te in s cou ld be s tu d ied w ith th is m e th o d b y in c reas in g or d ec rea s in g th e g e l p o ly m er co n c e n tra ­tio n , a n d d e c re a s in g o r in c re a s in g th e tim e o f e lec tro p h o resis . O f p a rticu la r in ­te re s t, IgM is in c lu d e d in th e h ig h e r m o lecu lar w e ig h t p ro te in m ixture.

Also in ta b le I is in c lu d e d a m ean v a lue an d s tan dard dev ia tion o f th e ca lcu la ted m o lecu lar w e igh ts o f th e te n d is tin g u ish ­ab le p ro te ins . T e n d iffe ren t norm al sub ­jec ts an d five d iffe ren t exp erim en ts w ere u se d in th e s e ca lcu la tio n s . T h e in itia l id en tifica tio n w as m ade on th e basis o f R f value an d qu an tity . T he id en tifica tio n of th e Ig G a n d a lb u m in b a n d s w as la te r su p p o rted by O u ch te rlo n y d o u b le diffu­sion. L ik ew ise , th e p re sen ce o f IgM n ea r th e to p o f th e ge l w as d em o n stra ted by th e O u ch te rlo n y tech n iq u e .

T h ree p a tien ts w ith abnorm al ce llu lo se

TABLE I

Bands Owing to Normal Human Serum Proteinsin the 70 to 250,000 Molecular Weight Range

UsualStandard Concentration

Molecular Deviation in Human SerumWeight N=10 Identification mg per dl

272,000 + 14,000258,000 + 4,000249,000 + 4,000236,000 + 10,000 (C4 Component) 20 to 50204,000 + 14,000 (High density

lipoproteins)290 to 770

178,000 + 10,000 (C3 Component IgA) 80 to 140156,000 + 17,000 IgG 800 to 1,800133,000 + 17,000 Albumin dimer101,000 + 10,000 (Transferrin, 200 to 400

haptoglobin) 100 to 30069,000 + Albumin 3,500 to 4,500

a ce ta te p ro te in e lec tro p h o resis an d ab ­n o rm al agarose im m u n o e le c tro p h o res is w ere cho sen for particu lar study.

In f ig u re 2 are sho w n th e re su lts o f S D S -po lyacry lam ide e lec tro p h o re s is o f th e seru m o f W. P. N o te th e heav y b an d (arrow) in th e reg ion o f m olecu lar w e ig h t 1 6 0 ,0 0 0 . T h is p a t ie n t h as a w e l l d o c u m e n te d m u l t ip le m y e lo m a . T h e p arap ro te in was show n by im m u n o elec­trop ho resis to be IgG , w ith k ty p e ligh t c h a in s . I t w as a lso a c ry s ta llin e c ry o ­g lobu lin .

In figu re 2 are sho w n th e re su lts o f SD S -gel e lec tro p h o resis o f th e p u rified cryog lo bu lin . N ote th e d o u b le b a n d in th e reg ion o f m olecu lar w e igh t 160,000. T he q u an tita tiv e ratio o f th e slow er m ov­ing b an d to th e faster was rep ro d u c ib ly ab o u t 2 :1 in six runs do n e at th e d iffe ren t t im e s from c ry o p re c ip i ta te s p r e p a r e d from th re e d iffe ren t serum sam ples. T he tw o b a n d s o f th e u n s ta in e d g e l w e re e lu te d separa te ly and show n to b e b o th IgG b y O u ch te rlo n y im m unodiffusion .

In f ig u re 3 are sho w n th e re su lts o f S D S -po lyacry lam ide e le c tro p h o re s is o f th e se ru m o f J. D . N ote th e bands a t th e to p o f th e g e l a n d in th e r e g io n o f m o lecu la r w e ig h t 160,000. L ikew ise , th e s e ru m o f th is p a t ie n t y ie ld e d a cryo- p re c ip ita te , sh o w n im m u n o lo g ica lly to b e m ixed IgG -IgM (figure 3).

In figu re 4 are sho w n th e re su lts o f e lec tro p h o resis o f serum from J. M. T h is p a tie n t h ad severe liver d isease accom ­p a n ie d b y p ro d u c tio n o f a m on oclo nal IgG . H is p a rap ro te in w as show n to b e IgG . H e also p ro d u ced a po lyclonal in ­c reased am o u n t o f IgA, as w ell as no rm al am ounts o f IgM.

In figu re 4 are also show n resu lts o f th e e lec tro p h o resis o f his co n cen tra ted u rin e . N ote th e b a n d (arrow) p ro d u ced by lig h t chain p ro te in . T his was show n im m u n o ­logically to b e o f k type.

T h e p o ly ac ry lam id e g e l e le c tro p h o ­re s is o f S D S c o m p le x e d s e ru m p ro -

1 8 2 G R IFFITH S, ET AL.

m

I

Figure 3. S tandard (left), an electrophoretogram o f the cryoprecip itate from th e serum o f J. D ., an d an electrophoretogram o f the serum o f J. D . (% th e usual concentration). T hese sam ples w ere analyzed during different experim ents and w ere run for d iffe ren t tim es. F igure 4. S tandard (left), and electrophoretogram s of the serum and urine (respectively) o f J. M.

te in s has p ro v en to b e a re p ro d u c ib le h igh reso lu tion te ch n iq u e in th e m olecu­lar w e ig h t range 70,000 to 300,000. It s e rv e s as a v a lu a b le a d ju n c t to im - m un oe lec tro ph oresis an d im m un od iffu ­sion as a tech n iq u e o f th e study o f para- p ro te inem ias. Its h igh re so lv in g po w er enabled the authors to dem onstrate a m ixed IgG -IgG cryog lobu lin . M ixed IgG -IgM cry o lo b u lin s have b e e n d e sc r ib e d an d ch a ra c te r iz e d im m u n o lo g ica lly .9 H o w ­ever, IgG -IgG m ixed cryog lobulin s are difficu lt to dem onstra te , since each p ro ­te in w ould have sim ilar im m unologic and chrom atographic p ro p e rtie s .1 It has b een re p o r te d th a t w h o le im m u n o g lo b u lin s w ill no t m igrate according to m olecular w e ig h t in th e S D S -po lyacry lam ide sys­te m .14 T he p roposed reason was th a t th e com plex te rtia ry s tructure o f th ese large m olecu les p reven ts effic ien t com plex ing o f the SDS. T he 4M urea ap p a ren tly c ir­c u m v en ts th is p ro b le m s in c e th e im ­

m unoglobulins do m igrate in accordance w ith theory in our system .R eferences

1. B a l e s t r i e r i , G., I n n e rn iz z i , F ., C o n so g n o , G., R o sso , V., S e c o n d o ,D . S., T in ian i, A., and ZANUSSI, C.: N ature o f the antigam m aglobulin activ ity in cryog lobu linem ic d isorders . Acta H aem at. 52:159-169, 1974.

2. B lo m b e rg , D. J. and B u rk e , M. D.: Isoen­zym es o f crea tin e k inase; separa tion by ac- rylam ide gel electrophoresis. Ann. C lin . Lab. Sci. 4:456-461, 1974.

3. C a w le y , L .: E le c tro p h o re s is an d Im ­m unoelectrophoresis. L ittle & Brown Boston, pp. 247-254, 1969.

4. D av is, B. J.: D isc electrophoresis-II. Ann N. Y. Acad. Sci. 121 :404-427, 1964.

5. D u n k e r, A. K. and R u e c k e r t , R. R.: Observa­tions on m olecular w eigh t determ inations on polyacrylam ide gel. J. Biol. C hem . 244:5074- 5080, 1969.

6. F e lg e n b a u e r , K.: Im m unoglobulins in disc electrophoresis. Clin. Chim . Acta 39:177-181,1972.

7. K a p la n , M. M. and R o g e r s , L.: Separation of h u m an se ru m -a lk a lin e -p h o sp h a ta se iso e n ­zymes by polyacrylam ide gel electrophoresis. Lancet 2:1029-1031. 1969.

ANALYSIS O F HUMAN SERUM 183

8. L e v in e , D .: M odel R101 M icro zo n e E le c ­trophoresis C ell Instruc tion M anual. Beckm an Instrum ents, F u llerton , CA, pp. 21-41, 1965.

9. M e l t z e r , M . a n d F r a n k l in , E . C .: C ry o g lo b u lin em ia— a s tu d y o f 29 p a tien ts . Amer. J. M ed. 40:828-836, 1966.

10. Or n STEIN, L.: D isc electrophoresis-I. Ann. N. Y. Acad. Sci. 121 :321-329, 1964.

11. Re y n o l d s , J. A. and T a n f o r d , C.: T he gross conform ation o f p ro tein-sodium dodecyl su l­fate com plexes. J. Biol. C hem . 245:5161-5165,1970.

12. Sh a p ir o , A. L., Vin n e l a , E., an d Ma iz e l , J.

V.: M olecular w eigh t estim ation o f po lypep­tid e c h a in s b y e le c tr o p h o re s is in SD S - polyacrylam ide gels, B iochem . Biophys. Res. Com m . 28:815-820, 1967.

13. T u n g , J. S. and Kn ig h t , C. A.: E ffect o f charge on th e determ ination o f m olecular w eig h t o f p ro te in s b y g e l e le c tro p h o re s is in SD S . B iochem . B iophys. Res. Com m . 4 2 :1117-1121,1971.

14. VlRELLA, G. and D e F r e it a s , M. M.: S tructural c h a ra c te r iz a tio n o f im m u n o g lo b u lin s c o n ­ta in ed in polyacrylam ide gels. E x perim en tia 29:142-144, 1973.