J.

Mario McDermott
November 6, 1998
CH 533-01
Dr. Olesen
SDS-PAGE Electrophoresis as a Method for Determining the Molecular Weight of
an Unknown Protein
Procedure:
!is e"#eriment $as an e"ercise on %eterminin& t!e molec'lar $ei&!ts o(
#roteins b) 'sin& *D*-+,-. electro#!oresis to se#arate t!e #roteins accor%in& to
molec'lar $ei&!t. ,n /MC &el 0112 #ol)acr)lami%e3 $as #lace% in t!e electro#!oresis
a##arat's alon& $it! 14 r'nnin& b'((er 054 r'nnin& b'((er %il'te% 155 $it! $ater3. !e
&el $as s!are% $it! anot!er t$o-#erson &ro'#, an% 5 l o( t!e stan%ar%, containin& 6
#roteins o( 7no$n molec'lar $ei&!ts, $ere a%%e% to lanes 1, 6, an% 11 0a(ter inc'batin&
in a boilin& $ater bat! (or abo't 8 min'tes3. One m& o( 'n7no$n #rotein 95 $as %il'te%
in 1 m: o( sam#le b'((er 0$!ic! containe% ris-HCl, &l)cerol, *D*, bromo#!enol bl'e,
an% -merca#toet!anol3. !e sol'tion $as $arme% in t!e boilin& $ater bat! (or 8
min'tes to %enat're t!e #roteins. !en, 5 l an% 10 l o( t!e 'n7no$n ; b'((er sol'tion
$ere a%%e% to lanes 3 an% 5, res#ectivel).
115 < $as a##lie% to t!e &el via a #o$er s'##l) 'ntil t!e bromo#!enol bl'e ban%
reac!e% t!e bottom e%&e o( t!e &el 0abo't 8.5 cm3. ,(ter removal (rom t!e
electro#!oresis a##arat's, t!e &el $as staine% 'sin& 0.152 Coomassie =l'e %)e in
met!anol5 acetic aci%5 $ater, 55155. ,(ter 15 min'tes o( stainin&, t!e %)e $as remove%,
an% t!e &el $as %estaine% 'sin& anot!er sol'tion 0met!anol5 acetic aci%5 $ater, 6565863
over a #erio% o( abo't 5-6 %a)s. !e %estain sol'tion $as re#lace% $!enever it a##eare%
sat'rate% $it! t!e %)e. !e &el $as t!en anal)>e% 'n%er a li&!t bo" an% t!e %istance to
eac! ban% (rom t!e $ells $as recor%e%.
Data:
Gel and positions measured from the wells for each standard !measured in cm":
#and Position Standard $ane %& Standard $ane %' Standard $ane %(&
1 1.11 1.90 1.93
1 3.05 3.05 1.55
3 3.30 3.31 1.95
8 3.93 3.91 3.15
5 No =an% No =an% 3.80
6 No =an% No =an% 3.80
Determination of )elati*e Distance +ra*eled , Each #and !-alculated )atio":
0!e i%enti(ication o( t!e a##ro#riate molec'lar $ei&!t $as %etermine% b) t!e
strai&!ter &ra#! t!at is attac!e% to t!is re#ort. =ot! &ra#!s $ere b) omittin& t!e !i&!est
molec'lar $ei&!t #rotein or t!e lo$est molec'lar $ei&!t #rotein (rom eit!er &ra#!. !e
&ra#! $it! t!e strai&!test relations!i# bet$een relative %istance an% lo& o( molec'lar
$ei&!t $as t!e &ra#! t!at omitte% t!e -lactalb'min3.
Protein Molecular
Wgt !in
Daltons"
Distance
+ra*eled ,
Protein !cm"
Distance +ra*eled
, #romophenol
#lue !cm"
-alculated
)atio
=ovine *er'm
,lb'min
66,000 1.93 8.50 0.818
.&& ,lb'min 85,000 1.55 8.50 0.566
-l)ceral%e!)%e-
3-+!os#!ate
36,000 1.95 8.50 0.655
Carbonic
,n!)%ro's
19,000 3.15 8.50 0.600
r)#sino&en 18,000 3.80 8.50 0.655
r)#sin
?n!ibitor
10,000 3.80 8.50 0.888
-lactalb'min 18,100 Not +resent 8.50 N@,
Unknown %. Distance +ra*eled !cm"
:ane 93 05 : o( sam#le3 1.68
:ane 95 010 : o( sam#le3 1.68
Graphs:
*ee attac!e% &ra#!s , an% =. -ra#! , s!o$s t!e relations!i# o( relative %istance
travele% vers's lo& o( molec'lar $ei&!t $it! t!e stan%ar% #rotein -lactalb'min omitte%.
!e tren%line o( t!e &ra#! is ver) close to t!e #lotte% #oints, m'c! closer t!an t!e
tren%line in -ra#! =, $!ic! is t!e same &ra#! i( bovine ser'm alb'minAs molec'lar
$ei&!t is omitte%. !is s!o$s t!at t!ere is a more linear relations!i# bet$een %istance
move% an% lo& o( molec'lar $ei&!t i( -lactalb'min is i%enti(ie% as t!e missin& #rotein
ban% on o'r &el.
!e molec'lar $ei&!t o( t!e 'n7no$n #rotein 0953 $as %etermine% to be 36,691
%altons (rom -ra#! , an% 19,080 %altons (rom t!e tren%line eB'ation in -ra#! =.
-onclusion:
/rom -ra#! ,, $e %etermine% t!e molec'lar $ei&!t o( 'n7no$n #rotein 95 to be
a##ro"imatel) 36,691 %altons. !is is vali% concl'sion beca'se $e %etermine% t!at t!e
%ata incl'%e% in -ra#! , !ave a more linear relations!i# t!an t!e %ata &ra#!e% in -ra#!
=. O'r best stan%ar% lane containe% onl) 6 o't o( 6 ban%s. !ere(ore, $e !a% to t!ro$
o't one o( t!e #roteins to %etermine t!e relations!i# o( t!e %istance travele% b) t!e ban%
to t!e lo& o( t!e molec'lar $ei&!t. ?n -ra#! ,, $e ass'me% t!at -lactalb'min $as ver)
li&!t in molec'lar $ei&!t an% ma) !ave r'n com#letel) o(( t!e &el an% omitte% it (rom t!e
calc'lations. !e tren%line o( t!is &ra#! lies closer to all o( t!e #oints t!an t!e tren%line
in -ra#! =, t!ere(ore, t!ere is a close correlation bet$een %istance move% an% lo& o(
molec'lar $ei&!t. ?n -ra#! =, bovine ser'm alb'min 0!i&!est molec'lar $ei&!t
stan%ar%3 $as ass'me% to !ave %i(('se% too m'c! %'rin& electro#!oresis to be seen in t!e
staine% &el. !e tren%line $as a strai&!t line t!at $as not ver) (ar (rom t!e %ata #oints,
b't $as not as close to t!e #oints as in -ra#! ,. !is $o'l% be a so'rce o( error i( t!e -
lactalb'min stan%ar% %i% not, in (act, r'n o(( t!e &el. ='t o'r %ata %oes s'&&est t!at t!is
is in%ee% t!e case.
,not!er #robable so'rce o( error $as o'r stainin& tec!niB'e. !e =io-Ca%
instr'ctions s'&&est stainin& in t!e 0.152 Coomassie =l'e (or abo't 30 min'tes, b't $e
onl) staine% (or 10 min'tes. O'r lo$ere% amo'nt o( stainin& #robabl) ca'se% (e$er
ban%s to s!o$ '# $!en meas'rin& t!e #rotein ban%s in t!e &el. ,lso, t!is ma) !ave
ma%e seein& t!e ban%s t!at $e %i% meas're a little easier. .rrors in meas'rin& t!e
%istance (rom t!e $ells to t!e ban%s ma) !ave occ'rre% beca'se t!e #rotein ban%s $ere
so (aint. ,lso, loa%in& ot!er stan%ar% lanes at 10 l alon& $it! t!e 5 l lanes co'l% !ave
also !el#e% to see t!e stan%ar% #rotein ban%s more clearl).
*D*-+,-. electro#!oresis 'ses molec'lar $ei&!t to se#arate to se#arate #roteins
across a #ol)acr)lami%e matri". *D* is 'se% to treat all o( t!e #roteins an% &ive t!em an
a##ro"imatel) eB'al ne&ative c!ar&e so t!at t!e #roteins are #'s!e%, b) t!e c'rrent, at
abo't t!e same rate. !is allo$s t!e ca'ses t!e #roteins to mi&rate t!ro'&! t!e &el
matri", to$ar% t!e #ositive electro%e. *maller #roteins move B'ic7l) t!ro'&! t!e #ores
in t!e cross-lin7e% #ol)acr)lami%e an% mi&rate (art!er t!an b'l7) #roteins $it! !i&!
molec'lar $ei&!ts. !is %i((erence in mi&ration rates ca'ses t!e small #roteins to be
se#arate% (rom t!e !i&!er molec'lar $ei&!t #roteins. Di((erent 2+,-. concentrations
re(er to t!e amo'nt o( cross-lin7in& o( t!e #ol)mers in t!e &el matri". D!en t!e
#ercenta&e is increase%, t!e si>e o( t!e #ores %ecreases. Hi&!er 2+,-. &els resolve
lo$er molec'lar $ei&!t #roteins ver) e((ectivel), b't cannot %istin&'is! amon& !i&!
molec'lar $ei&!t #roteins beca'se t!e) %o not move ver) (ar t!ro'&! t!e &el. :o$er
2+,-. &els allo$ lar&er #roteins to travel a &oo% %istance a$a) (rom t!e $ells, $!ic!
&ives a &reater resol'tion o( !i&!er molec'lar $ei&!t #roteins. :o$ molec'lar $ei&!t
#roteins are #rone to bein& r'n o(( t!e &el $!en resolvin& lar&er #roteins in a lo$
2+,-. &el.