Guía TP Proteínas

8/18/2019 Guía TP Proteínas

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FACULTAD CIENCIAS EXACTAS NATURALES Y AGRIMENSURA - UNIVERSIDAD NACIONAL DEL NORDESTECARRERA BIOQUIMICA - AREA BIOQUÍMICA CLINICA - ASIGNATURA QUÍMICA CLINICAJTP CEI Versión en r!"es!

GUIA DE TRABAJO PRACTICO - LABORATORIO

PROTEINAS SERICASO#$e%i&!s De%er'in("ión )e r!%e*n(s %!%(+es !r +( re(""ión )e+ #i,re%

De%er'in("ión )e (+#.'in( !r ,nión (+ "!+!r(n%e Ver)e )e Br!'!"res!+ Re(+i/("ión e in%erre%("ión )e ,n( "!rri)( e+e"%r!0!r1%i"( en ("e%(%! )e "e+,+!s( 2 &(+!r("ión

",(+i%(%i&( )e +(s r!%e*n(s s1ri"(s

ETAPA PREANALITICA34 M,es%r(L( ',es%r( )e e+e""ión (r( +( )e%er'in("ión )e r!%e*n(s %!%(+es 2 r!%ein!5r('( e+e"%r!0!r1%i"! ess,er! L(s r!%e*n(s %!%(+es )e+ +(s'( s!n 67 58)+ '(s 9,e en s,er! !r +( resen"i( )e+ 0i#rinó5en!P(r( +( )e%er'in("ión )e (+#.'in( se ,e)e ,%i+i/(r in)is%in%('en%e s,er! , +(s'(L!s s,er!s "!n +ie'i( 28! :e'ó+isis '!)er()( ! se&er( ;<# =6> 58)+4 n! )e#en r!"es(rse L!ss,er!s "!n #i+irr,#in( %!%(+ in0eri!r ( 36 '58)+ ,e)en r!"es(rse C!nser&("ión )e +( ',es%r(

L(s r!%e*n(s %!%(+es )e+ s,er! s!n es%(#+es ),r(n%e 3 se'(n( ( %e'er(%,r( ('#ien%e 2 3 'es ",(n)!se re0ri5er(n A ?>6 @C +(s r!%e*n(s ),r(n > 'eses 2 +( (+#.'in( > (!s P(r( re(+i/(r e+e"%r!0!resis,e)e re0ri5er(se e+ s,er! :(s%( :s <!r( )e ',es%re! e in5es%( re"ien%e )e (+i'en%!s

N! se :(n !#ser&()! &(ri("i!nes )i,rn(s en +( "!n"en%r("ión )e r!%e*n(s s1ri"(s ni !r in5es%(re"ien%e )e (+i'en%!s Es%(sis &en!s!

L( e+e&("ión )e PT 2 (+#.'in( !r es%(sis &en!s! )een)en )e+ 5r()! 2 ),r("ión )e+ 'is'! 'ien%r(s9,e +!s !r"en%($es )e +( e+e"%r!0!resis n! se (+%er(n De#e 'ini'i/(rse e+ es%(sis &en!s! ),r(n%e +(&en!,n"ión C('#i!s )e !s%,r(

En +!s in)i&i),!s n!r'(+es +( "!n"en%r("ión )e r!%e*n(s (,'en%( ( 36 ",(n)! (s(n )e +(!si"ión ("!s%()( ( +( )e ie Es%!s "('#i!s )e "!n"en%r("ión r!%ei"( )e#i)!s ( "('#i!s )e+ (5,(+(s'%i"( se r!),"en en 3 'in,%!s E$er"i"i!

En +(s :!r(s si5,ien%es (+ e$er"i"i! in%ens! ,e)e :(#er '!)er()( )es:i)r(%("ión eH%r("e+,+(r 2 "!ne++! :e'!"!n"en%r("ión 9,e e+e&( +( r!%eine'i( E+ e0e"%! (,'en%( ( '(2!r %e'er(%,r( ('#ien%e 2ni&e+ )e e$er"i"i! E+ (,'en%! 9,e se re5is%r( es )e '(5ni%,) se'e$(n%e ( +!s "('#i!s "(,s()!s !r +(!s%,r( 2 re9,iere 76 'in,%!s (r( &!+&er ( +!s &(+!res #(s(+es Us! )e (n%i"!n"e%i&!s 2 e'#(r(/!

E+ ,s! )e (n%i"!n"e%i&!s !r(+es 2 e+ e'#(r(/! )is'in,2en +( "!n"en%r("ión s1ri"( )e r!%e*n(s %!%(+esrin"i(+'en%e )e#i)! ( )is'in,"i!nes )e (+#.'in( L(s 0r(""i!nes r!%ei"(s α3 α> 2 β (,'en%(n+i5er('en%e "!n e+ ,s! )e (n%i"!n"e%i&!s !r(+es 2 &,e+&en ( ser n!r'(+es ( +(s 3> se'(n(s )e "es(r )i":! ,s!

In%er0eren"i(s !r 'e)i"('en%!sIn%er0eren"i(s (n(+*%i"(sEn e+ )!s($e )e (+#.'in( "!n &er)e )e #r!'!"res!+ e+ "i)! ("e%i+s(+i"i+i"! +( "(r#eni"i+in( 2 +("e0!%(Hi'( (s* "!'! +( :e(rin( ,s()( in &i%r! "(,s(n 0(+s!s )es"ens!s L( ('i"i+in( ( )!sise+e&()(s r!),"e 0(+s!s (,'en%!s L( eni"i+in( r!),"e ,n )es)!#+('ien%! )e +( (+#.'in( en +(e+e"%r!0!resis s!#re "e+,+!s( ! (5(r!s(Se "!n!"e ,n( ('+i( 5('( )e 'e)i"('en%!s 9,e ( )!sis :(#i%,(+es n! in%er0iereIn%er0eren"i(s 0(r'("!+ó5i"(sL(s er0,si!nes in%r(&en!s(s r!),"en )is'in,"i!nes )e +( (+#,'ine'i( en ese"i(+ si se ,%i+i/(n

eH(ns!res "!'! )eH%r(n! ! '(ni%!+ L!s )i,r1%i"!s en ese"i(+ +( 0,r!se'i)( r!),"en e+e&("i!nes!r :e'!"!n"en%r("ión

3




L!s es%ró5en!s r!&!"(n )es"ens! )e +( (+#,'ine'i( !r in:i#i"ión )e s, s*n%esis :e%i"( L(s )!sis:(#i%,(+es )e > ,58)*( ,s()(s en ',$eres !s'en!,si"(s "(,s(n s!+! +e&es )es"ens!s L!sr!5es%5en!s n! (+%er(n +( (+#,'ine'i(L( (s(r(5in(s( ,s()( en :e'!(%*(s "(,s( )es"ens!s )e :(s%( >> +,e5! )e se'(n(sL( )(s!n( ,n (n%i+er!s! %r(s %r(%('ien%! r!+!n5()! r!),"e )es"ens!s )e :(s%( 6 !r (,'en%!)e+ "(%(#!+is'! )e +( (+#.'in(

Me)i"('en%!s :e(%!%óHi"!s en %r(%('ien%!s r!+!n5()!s "(,s(n )es"ens!s )e (+#.'in( !r )is'in,"ión )e s, s*n%esis

ETAPA ANALITICA34 Me%!)!+!5*( Cuantificación de Proteínas Totales "METODO DEL BIRET"FUNDAMENTOE+ '1%!)! se )es(rr!++ó ( (r%ir )e +( !#ser&("ión )e 9,e e+ #i,re% )(#( ,n "!'+e$! )e "!+!r .r,r( (+re(""i!n(r "!n ,n( s!+,"ión (+"(+in( re5,+()( )e s,+0(%! )e "!#re L(s r!%e*n(s 2 (+5,n(s ('in(sre(""i!n(n )e 0!r'( se'e$(n%e L!s i!nes "!#re 0!r'(n ,n "!'+e$! )e "!!r)in("ión "!n ",(%r! 5r,!s-N< n,"+e!0*+i"!s 9,e en +( re(""ión "!n +(s r!%e*n(s r!&ienen )e +!s en+("es e%*)i"!s )e +!s('in!"i)!s E+ "!'+e$! ',es%r( 'Hi'!s )e (#s!r"ión ( 776 2 6 n' L( (#s!r#(n"i(5ener(+'en%e se 'i)e ( 6 n' ,es (,n9,e es '(2!r +( sensi#i+i)() ( 776 n' es 's 0"i+ 9,e se

resen%en in%er0eren"i(s L( (#s!r#(n"i( 'e)i)( ( 6 n' es )ire"%('en%e r!!r"i!n(+ ( +("!n"en%r("ión )e r!%e*n(s %!%(+es en +( ',es%r(E+ re("%i&! )e #i,re% es% 0!r'()! !r ,n( s!+,"ión (+"(+in( )e s,+0(%! )e "!#re ( +( 9,e se (()e%(r%r(%! só)i"!-!%si"! (r( e&i%(r 9,e re"ii%en +!s i!nes ".ri"!s en 0!r'( )e :i)róHi)! A &e"es se(()e i!),r! !%si"! (r( e&i%(r +( re),""ión es!n%ne( )e +!s i!nes ".ri"!s (,n9,e n! s,e+e ser ne"es(ri! ",(n)! e+ s,+0(%! )e "!#re es ,r! 2 se ,%i+i/(n "!n"en%r("i!nes ()e",()(s )e %(r%r(%!

TECNICA OPERATORIAM,es%r( S,er! +i#re )e :e'ó+isis

M(%eri(+ re9,eri)! Ese"%r!0!%ó'e%r! ! F!%!"!+!r*'e%r! Mi"r!ie%(s 2 ie%(s "(("es )e 'e)ir +!s &!+.'enes in)i"()!s T,#!s )e ens(2! T,#!s )e 0!%!"!+!ri'e%r! ! ",#e%(s ese"%r!0!%!'1%ri"(s B(! )e (5,( ( 7 KC Re+!$ ! Ti'er

Re("%i&!s Tiene )i0eren"i(s se5.n e+ 0(#ri"(n%e Re("%i&! C,r!(+"(+in! GT L(# iener Bi!s2s%e's

S,+0(%! )e C!#re 3> 'M 37 'M 'M T(r%r(%! só)i"!-!%si"! 7> 'M I!),r! !%si"! 'M <i)róHi)! só)i"! >6 'M 'M 336'M

E+ R&! iener %iene EDTA 2 (+9,i+(ri+!+ie%er )e "!n"en%r("ión n! )e"+(r()(E+ R&! Bi!s2s%e's %iene ,n )e%er5en%e sin ese"i0i"(r s, "!'!si"ión 2 "!n"en%r("iónE+ re("%i&! ",r!(+"(+in! )e#e "!nser&(rse ( %e'er(%,r( ('#ien%e en +,5(r 0res"! 2 (+ (#ri5! )e +( +,/Es "!rr!si&!

C(+i#r()!r S!+,"ión )e (+#.'in( 2 5+!#,+in(s en es%()! n(%i&! )e "!n"en%r("ión )e%er'in()( 0ren%e (+es%n)(r )e re0eren"i( )e+ N(%i!n(+ Ins%i%,%e !0 S%(n)(r%s (n) Te":n!+!5:2 SRM ># C!nser&(r ( >-@C :(s%( s, 0e":( )e &en"i'ien%!P,e)e ,%i+i/(rse ",(+9,ier "(+i#r()!r "!'er"i(+ )e '(%ri/ r!%ei"( sie're 9,e %en5( %r(/(#i+i)() "!nes%e es%n)(r ri'(ri!S,er! "!n%r!+ P!r e$e'+! Pre"in!r' ;L(# R!":e eH B!e:rin5er4V(+!r 'e)i! >> 58)+ 2 r(n5! ("e%(#+e ;8- >DS4 6 ? 58)+V(+!res !#%eni)!s 0ren%e (+ 'is'! es%n)(r ri'(ri! ;SRM >#4P,e)e ,%i+i/(rse ",(+9,ier s,er! "!n%r!+ "!'er"i(+ )e '(%ri/ r!%ei"( sie're 9,e %en5( %r(/(#i+i)() "!nes%e es%n)(r ri'(ri!

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PROCEDIMIENTOEn %,#!s r!%,+()!s "!+!"(r B+(n"! Tes%i5! C!n%r!+ M,es%r(Tes%i5! )e PT 6 ,+S,er! 6 ,+ 6 ,+

R&! C,r!(+"(+in! > '+ > '+ > '+ > '+Me/"+(r In",#(r en #(! )e (5,( ( 7 KC !r 3 'in,%!s Leer en ese"%r!0!%ó'e%r! ( 6 n' !0!%!"!+!ri'e%r! ( >6-6 n' ++e&(n)! ( "er! "!n re("%i&! )e %r(#($! C!+!r es%(#+e > :!r(sC+",+!s 0("%!r )e "+",+! 58)+ )e PT )e+ Tes%i5! A#s!r#(n"i( )e+ Tes%i5!

58)+ )e PT en e+ s,er! A#s!r#(n"i( )e +( M,es%r( H 0("%!r )e "+",+!

PERFORMANCE DEL METODO ;A,%!'(%i/(#+e4LINEALIDAD C,'+e "!n +( +e2 )e L('#er%-Beer :(s%( 3 58)+ ;GT 2 Bi!s2s%e's4 2 :(s%( 3> 58)+

;iener4 De%er'in(r e+ +*'i%e "!n e+ ins%r,'en%(+ )e +e"%,r( ( ,s(rESTABILIDAD E+ "!+!r (+"(n/( s, in%ensi)() 'Hi'( ( +!s 3 'in,%!s es%(#+e 3-> :!r(sLÍMITE DE DETECCIN T!)(s +(s r!%e*n(s re(""i!n(n )e ,n( 0!r'( si'i+(r '!s%r(n)! )i0eren"i(s',2 e9,e(s en%re "()( ,n( )e e++(s P(r( ,n "('#i! )e 6663 ,ni)()es )e (#s!r#(n"i( se re9,iere,n "('#i! '*ni'! )e 66> 58)+REPRODUCIBILIDAD INTRALABORATORIO INTERENSAYO A+ ni&e+ )e 58)+ se !#%,&! ,n"!e0i"ien%e )e &(ri("ión )e 7 (

V(ri("i!nes 0isi!+ó5i"(s A,'en%!s !r es%()!s )e )es:i)r(%("ión Dis'in,"i!nes !r es%()!s )es!#re:i)r(%("ión re%en"ión )e +*9,i)! 2 e'#(r(/! en 7er %ri'es%reV(ri("i!nes (%!+ó5i"(s N! %iene sen%i)! "!nsi)er(r+(s sin %ener e+ r!%ein!5r('( e+e"%r!0!r1%i"! )()!9,e +( "!rre+("ión "+*ni"( se re(+i/( #(sn)!se en +(s &(ri("i!nes !#ser&()(s en +(s 0r(""i!nes )e+

'is'!

>4 Me%!)!+!5*( Cuantificación de Al!#ina $M%todo de &i'ación al (erde de Bro#ocresol)FUNDAMENTOL( (+#.'in( %iene ,n( (+%( (0ini)() !r (ni!nes !r5ni"!s "!+!r(n%es "!n +( ,nión se e+e&('(r"()('en%e e+ i"! )e (#s!r"ión )e+ "!+!r(n%e 2 )()! 9,e es%e in"re'en%! es r!!r"i!n(+ ( +("(n%i)() )e (+#.'in( ,e)e ",(n%i0i"(rse +( 'is'(C!n es%e rin"ii! se ,%i+i/(n +!s "!+!r(n%es .r,r( )e #r!'!"res!+ &er)e )e #r!'!"res!+ 2 (/,+ )e#r!'!0en!+ L( (+#.'in( 2 e+ &er)e )e #r!'!"res!+ se ,nen ins%(n%ne('en%e ( < en%re 7 2 > enresen"i( )e ,n( ()e",()( 0,er/( ióni"( )(n)! +,5(r ( ,n 5r(n (,'en%! )e +( (#s!r#(n"i( ( > n'rese"%! )e +( s!+,"ión )e+ "!+!r(n%e +i#re Es%e in"re'en%! es r!!r"i!n(+ ( +( "!n"en%r("ión )e

(+#.'in( en +( ',es%r( E+ "!+!r(n%e es )e ,n "!+!r ('(ri++! 'ien%r(s 9,e e+ "!'+e$! r!%e*n(-"!+!r(n%e %iene ,n in%ens! "!+!r (/,+ &er)!s! L( (+%( (0ini)() (+#.'in( - "!+!r(n%e :("e 9,e %!)( +((+#.'in( se ,n( en 'en!s )e 76 se5,n)!s )e ser 'e/"+()!s Se (5re5( ,n )e%er5en%e n! ióni"! (r(e&i%(r en%,r#i('ien%! 2 #($(r +( (#s!r#(n"i( )e+ re("%i&! 2 "+!r,r! )e s!)i! (r( )is'in,ir +( in%er0eren"i(!r 5+!#,+in(s

TECNICA OPERATORIAM,es%r( S,er! ! +(s'( +i#re )e :e'ó+isis e i"%eri"i(

M(%eri(+ re9,eri)! Ese"%r!0!%ó'e%r! ! 0!%!"!+!ri'e%r! Mi"r!ie%(s 2 ie%(s "(("es )e 'e)ir +!s &!+.'enes in)i"()!s

T,#!s )e ens(2! Re+!$ ! %i'er

Re("%i&!s Ver)e )e Br!'! Cres!+ ;VBC4 ! #r!'!"res!+s,+0!n0%(+ein( ;BCF4

7




L( "!n"en%r("ión )e VBC &(r*( ',":! se5.n e+ 0(#ri"(n%e iener ;766 ,M4 GTL(# ;6 ,M4Bi!s2s%e's ;36 ,M4 L( "!n"en%r("ión re"!'en)()( (r( 'Hi'( +ine(+i)() es )e 6 ,M ;(+(n -Pes"e4 B,00er S,""in(%! 'M < 7 Tensi!("%i&! n! ióni"! !+i!Hie%i+en +(,ri+ 1%er (+ 3 C+!r,r!)e s!)i! 6 MEs%e re("%i&! )e#e "!nser&(rse ( %e'er(%,r( ('#ien%e en +,5(r 0res"! 2 (+ (#ri5! )e +( +,/

C(+i#r()!r S!+,"ión )e (+#.'in( 2 5+!#,+in(s en es%()! n(%i&! )e "!n"en%r("ión )e%er'in()( 0ren%e (+es%n)(r )e re0eren"i( )e+ N(%i!n(+ Ins%i%,%e !0 S%(n)(r%s (n) Te":n!+!5:2 SRM ># C!nser&(r ( >-@C :(s%( s, 0e":( )e &en"i'ien%! P,e)e ,%i+i/(rse ",(+9,ier "(+i#r()!r "!'er"i(+ sie're 9,e %en5(%r(/(#i+i)() "!n es%e es%n)(r ri'(ri!S,er! "!n%r!+ P!r e$e'+! Pre"in!r' ;L(# R!":e eH B!e:rin5er4V(+!r 'e)i! 7 58)+ 2 r(n5! ("e%(#+e ;8->DS4 76 ? 73 58)+V(+!res !#%eni)!s 0ren%e (+ 'is'! es%n)(r ri'(ri! ;SRM >#4P,e)e ,%i+i/(rse ",(+9,ier "(+i#r()!r "!'er"i(+ sie're 9,e %en5( %r(/(#i+i)() "!n es%e es%n)(r ri'(ri!

PROCEDIMIENTO

Tr(#($(r en%re 3 2 > @C A$,s%(r e+ ese"%r!0!%ó'e%r! ( > n' ! 0!%!"!+!r*'e%r! ( >6-6 n'++e&(n)! ( "er! "!n re("%i&! )e %r(#($!En %,#!s r!%,+()!s "!+!"(r B+(n"! Tes%i5! C!n%r!+ M,es%r(Tes%i5! )e A+#.'in( 36 ,+S,er! ! +(s'( 36 ,+ 36 ,+R&! VBC 7 '+ 7 '+ 7 '+ 7 '+Me/"+(r !r in&ersión s,(&e Leer )en%r! )e +!s 3 ( 76 se5,n)!s (r( e&i%(r +( in%er0eren"i( !si%i&( )e+(s )e's r!%e*n(s +(s'%i"(s P!r e$e'+! +( "er,+!+(s'in( 2 e+ !r!s!',"!i)e re(""i!n(n "!n e+VBC ( +!s 'in,%!sC+",+!s 0("%!r )e "+",+! 58)+ )e A+#.'in( )e+ Tes%i5!

A#s!r#(n"i( )e+ Tes%i5!

58)+ )e A+#.'in( A#s!r#(n"i( )e +( M,es%r( H 0("%!r )e "+",+!

PERFORMANCE DEL METODOESPECIFICIDAD E+e&()( ",(n)! se re(+i/( +( +e"%,r( ( +!s 76 se5,n)!s )e +( 'e/"+( Es '(2!r 0ren%e(+ .r,r( )e #r!'!"res!+In%er0eren"i(s en)ó5en(s N! ,e)en ,%i+i/(rse s,er!s i"%1ri"!s "!n #i+irr,#in( = >6 '58)+ 28! "!n:e'ó+isis '!)er()( ! '(2!r 28! +ie'i( '(r"()( L!s %res in%er0ieren ne5(%i&('en%e L( :e(rin(r!),"e ,n( %,r#i)e/ 9,e !"(si!n( 0(+s!s (,'en%!s

LINEALIDAD C,'+e "!n +( +e2 )e L('#er%-Beer :(s%( 58)+ ;GTL(# Bi!s2s%e's 2 iener4 Le&es&(ri("i!nes se5.n e+ ins%r,'en%(+ )e +e"%,r(ESTABILIDAD E+ "!+!r (+"(n/( s, in%ensi)() 'Hi'( ( +!s >6 ( 76 se5,n)!s +,e5! &( (,'en%(n)!+en%('en%e :(s%( +!s >6 'in,%!s en 9,e "!'ien/( ( )e"re"erLIMITE DE DETECCION P(r( ,n "('#i! )e 6663 ,ni)()es )e (#s!r#(n"i( se re9,iere ,n "('#i!'*ni'! )e 663 58)+ )e (+#.'in(REPRODUCIBILIDAD INTRALABORATORIO INTERDÍAS A+ ni&e+ )e 7 58)+ se !#%iene ,n CV )e > (7

M1%!)! )e re0eren"i(<( si)! r!,es%! e+ e+e"%r!in',n!(n+isis e+ ",(+ se #(s( en +( 'i5r("ión )e +(s r!%e*n(s )e+ s,er!!r ,n "('! e+1"%ri"! en ,n 'e)i! "!n (5(r!s( "!n%enien)! (n%i(+#.'in( :,'(n( L( (+%,r( )e+ i"!

)e re"ii%("ión es r!!r"i!n(+ ( +( "!n"en%r("ión )e (+#.'in( en e+ s,er! Presen%( e+e&()(ese"i0i"i)() 2 re",er("ión 2 ("e%(#+e re"isión O%r!s '1%!)!s in',n!9,*'i"!s )e (+%(er0!r'(n"e s!n In',n!)i0,sión r()i(+ In',n!%,r#i)i'e%r*( In',n!ne0e+!'e%r*( R()i!in',n!(n+isis




2 En/i'!in',n!(n+isis T!)!s e++!s %ienen +( +i'i%("ión )e+ "!s%! 2 +( eHi5en"i( )e (+%( "(+i)() )e+(n%i",er! ,%i+i/()!E+ '1%!)! "!n .r,r( )e #r!'!"res!+ '!s%ró 'e$!r "!rre+("ión "!n e+ e+e"%r!in',n!(n+isis 9,e e+'1%!)! "!n VBC

In%er&(+! )e re0eren"i( (r( A+#.'in( en s,er!

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PROCEDIMIENTO




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