Centro Centro de Bachillerato Tecnológico Tecnológico e Industrial y de Servicios 108
!;<=/=, 3A4A LA /%<%4!>&AC>?& /% 3=4<%@&A, %& L=, AL>!%&<=,. AL>!%&<=,. Laboratorista Químico 5° “B” Laboratorista Analiza muestras de alimentos y bebidas alcohólicas con base a normas. Catedrático !i"uel #n"el $rancisco %duardo A"uilar Cruz &o.' Andrea (uadalu)e A"uilar (ómez &o.5 *+ctor %duardo Cansino ,oto &o.-' $atima Aleandra /íaz !azarie"os &o.-0 Carolina *ernández Bermudez &o.12 ,inar 3ascacio 4oas &o.' ,onia 6a7ueline 8elasco Castro &o.'0 3ablo 4amón 8entura ,uazna9ar &o.':
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Metodo de Kjeldahl INTR!"CCI#N $n el tra%ajo de rutina se deter&ina &ucho &'s (recuente&ente la )rote*na total otal +ue +ue las )rot )rote* e*na nass o a&i a&ino'c no'ciidos dos indi ndividu vidual ales es,, $n gene genera rall- el )rocedi&iento de re(erencia Kjeldahl deter&ina la &ateria nitrogenada total+ue incluye tanto las no )rote*nas co&o las )rote*nas verdaderas ./urand et al- 182 $l &3todo se %asa en la deter&inación de la cantidad de Nitrógeno org'n org'nic ico o cont conten enid ido o en )rod )roduc ucto toss ali& ali&en enta tari rios os-- co&)r co&)ro& o&et ete e dos dos )aso )asoss conse onseccuti utivos4 vos4 a2 5a desc desco& o&)o )osi sici ció ón de la &ate &ateri ria a org' org'ni nica ca %aj %ajo calenta&iento en )resencia de 'cido sul(6rico concentrado, %2 $l registro de la cantidad de a&oniaco o%tenida de la &uestra M/T$RI/5$SM/T$RI/5$S- $7"IS 9 R$/CTI:S o o o o o o o o
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1 &atra; Kjeldhal 1 tra&)a de va)or .Kjeldhal2 1 &atra; $rlen&eyer <00&l = es)'tulas 1 )i)eta de 10&l 1 jeringa .)i)etero2 $+ui)o necesario )ara titular Mangueras )ara el e+ui)o Kjeldhal erlas de e%ullición a)el tornasol >cido sul(6rico concentrado
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?idró@ido de sodio al A0 inc granulado Sul(ato de co%re )entahidratado Sul(ato de sodio anhidro >cido clorh*drico 0,1N >cido %órico al = araDna Rojo de &etilo )re)arado /;ul de &etileno )re)arado Balan;a anal*tica
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Indicador Shiro Tashiro4 Tashiro4 Me;clar = )artes de rojo de &etilo &e tilo y una de a;ul de &etileno,
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RC$!IMI$NT
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1, !eter&inar !eter&inar la &asa&asa- en la %alan;a anal*tic anal*ticaa- de a)ro@i&ada& a)ro@i&ada&ente ente un gra&o de &uestra y )asarla a un &atra; Kjeldahl- aEadirle = g de sul(ato de co%re- 10 g de sul(ato de sodio anhidro- =<&l de 'cido sul(6rico concentrado y unas )erlas de e%ullición, =, Colocar Colocar el &atra; &atra; en el digestor digestor y calentar calentar cuidadosa cuidadosa&ente &ente a %aja %aja te&)eratura hasta +ue todo el &aterial est3 car%oni;ado- cada 10 &inutos se de%er' girar el &atra;, F, /u&entar /u&entar gradual&ente gradual&ente la la te&)eratura te&)eratura hasta hasta +ue la disoluci disolución ón est3 co&)leta&ente clara y dejar )or F0 &inutos &'s a esa te&)eratura, A, $n(riar y aEadir aEadir de =00 =00 a F00&l de agua agua )ara disolver disolver co&)leta& co&)leta&ente ente la &uestra- agregar F ó A gr'nulos de ;inc- un )oco de )araDna cuando sea necesario .&uy )oca2 y <0&l de hidró@ido de sodio al A0, <, In&ediata&ent In&ediata&ente e conectar conectar el &atra; a un siste&a siste&a de destilacióndestilación- el cual cual )revia&ente se le ha colocado en la salida del re(rigerante un &atra; $rlen&eyer de <00&l +ue contenga <0&l de 'cido %órico y unas gotas del reactivo Shiro Tashiro co&o indicador, G, !estilar !estilar hasta +ue +ue haya )asado )asado todo el a&oniacoa&oniaco- +ue +ue unas gotas gotas de destilado no den alcalinidad con el )a)el )a )el tornasol- a)ro@i&ada&ente F00&l, NT/4 NT/4 5as )ri&eras gotas de destilado des tilado de%en hacer virar el color del indicador de violeta a verde, , Retirar Retirar el &atra; reci%ido reci%idorr y titular titular el destilado destilado con 'cido clorh*dri clorh*drico co 0,1 N o
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$HR$SI#N !$ R$S"5T/!S
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$l Nitrógeno )resente en la &uestra- e@)resado en )or ciento se
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$n donde4 : :olu&en de 'cido clorh*drico e&)leado en la titulación en &l
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N Nor&alidad del de l 'cido clorh*drico,
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& Masa de la &uestra en g, 0,01A Milie+uivalente del nitrógeno,
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$l )or ciento de )rote*nas se o%tiene &ulti)licando el )or ciento de nitrógeno o%tenido )or el (actor corres)ondiente
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$l contenido de nitrógeno en di(erentes )rote*nas es a)ro@i&ada&ente de 1G )or lo +ue &ulti)licando el )or ciento de nitrógeno o%tenido )or el (actor G,=< se o%tiene la cantidad de )rote*nas )resentes en el ali&ento,
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Metodo de 5oJry
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"N!/M$NT T$#RIC $l &3todo de 5oJryL .1<12 es un &3todo colori&3trico de valoración cuantitativa de las )rote*nas, / la &uestra se aEade un reactivo +ue (or&a un co&)lejo coloreado con las )rote*nas- siendo la intensidad de color )ro)orcional a la concentración de )rote*nas- seg6n la ley de 5a&%ert 5a&%ertBe Beer er / ,l,c ,l,c $ste $ste &3to &3todo do consta consta de dos eta)as eta)as44 12 5os iones Cu=O- en &edio alcalino- se unen a las )rote*nas (or&ando co&)lejos con los 'to&os de nitrógeno de los enlaces )e)t*dicos, $stos co&)lejos Cu=O)rote*na tienen un color a;ul claro, /de&'s- )rovocan el desd desdo%l o%la& a&ie ient nto o de la estr estruc uctur tura a trid tridi& i&ens ensio iona nall de la )rot )rote* e*na na-e@)oni3ndose los residuos (enólicos de tirosina +ue van a )artici)ar en la segunda eta)a de la reacción, $l Cu=O se &antiene en solución alca alcali lina na en (or& (or&a a de su co&) co&)le lejo jo con tartra tartrato to,, =2 5a redu reducc cció iónnta&%i3n en &edio %'sico- del reactivo de olinCiocalteau- )or los gru)os (enólicos de los residuos de tirosina- )resentes en la &ayor*a de las las )rot )rote* e*na nass- actu actuan ando do el co%r co%re e co&o co&o cata catali li;a ;ado dorr, $l )rin )rinci ci)a )all sti stit t d l ti d li Ci alt alt el 'ci 'cid
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Reacción entre la )rote*na y los reactivos
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=, M/T$RI/5$S 9 R$/CTI:S
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Tu%os Tu%os de ensayo Tu%os Tu%os alcon alcon .<0 &52 i)etas Color*&etro Cu%etas de color*&etro gitadores de tu%os
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Reactivo /4 Na=CF al =- Na? 0-1 M Reactivo B14 B14 CuSA P = al 1 Reactivo Reactivo B=4 tartrato sódico)ot'sico al = Reactivo Reactivo C4 Se )re)ara)re)ara- &e;clando &e;clando los reactivos4 reactivos4 /- B1 y B=- en )ro)orci )ro)orciones ones <040-<40-< .en volu&en2 Reactivo olinCiocalteau4 olinCiocalteau4 reactivo co&ercial diluido a 1QA Solución )atrón de al%6&ina de suero %ovino .BS/2 .= &gQ&52 Muestra )ro%le&a
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F, RC$!IMI$NT $H$RIM$NT/5 $H$RIM$NT/5 ara deter&inar la concentración de )rote*nas de la &uestra )ro%le&a se construye una curva )atrón o de cali%rado a )artir de una solución )atrón .BS/2 .= &gQ&52, 5a concentración +ue tienen las &uestras )ro%le&a se deter&ina )or inter)olación de los valores de a%sor%ancia en la curva )atrón, $l tu%o 0- +ue sólo contiene agua destilada y los reactivos- sirve de %lanco )ara el ajuste del color*&etro a cero de a%sor%ancia, asos a seguir4
1, Nu&erar del 0 al G- tu%os tu%os de de )l'stico )l'stico de de 10 Ml =, i)e i)ete tear ar las las cant cantid idad ades es de agua agua-- solu soluci ción ón )atr )atrón ón de al%6 al%6&i &ina na y solución )ro%le&a seEaladas en la ta%la, F, re)arar re)arar el reacti reactivo vo C- a )artir )artir de //- B1 y B= A, i)et i)etear ear a todos todos los tu%os tu%os el react reactivo ivo C, Me;clar Me;clar el conteni contenido do de cada tu%o y dejarlo re)osar 1< &inutos en oscuridad .dentro de la ta+uilla2, <, / contin continuac uación ión aEadir aEadir a todos todos los tu%os tu%os el react reactivo ivo de olin olin .dilui .diluido do 1QA2- &e;clando %ien )or agitación, !ejar re)osar F0 &inutos en la oscu oscuri rida dad d )ara )ara +ue +ue as* se desa desarr rrol olle le co&) co&)le leta ta&en &ente te la reac reacci ción ón
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)uesto +ue se resta resta el color de%ido de%ido a los reactivos reactivos,, /notar la &edida &edida de a%sor%ancia en la ta%la, o
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M3todo de Brad(ord
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"N!/M$NT Se %asa en la unión de un colorante- Co&assie Blue =<0 .ta&%i3n Serva Blue2 a las )rote*nas, $l colorante- en solución 'cida- e@iste en dos (or&as una a;ul y otra naranja, 5as )rote*nas se unen a la (or&a a;ul )ara (or&ar un co&)lejo )rote*nacolorante con un coeDciente de e@tinción &ayor +ue el colorante li%re, $ste &3todo es sensi%le .11< Ug2Ug2- si&) si&)le le-- r')i r')ido do-- %ara %arato to y )oca )ocass sust sustan anci cias as inte interD rDer eren en en su det deter&i er&ina naci ción ón,, $ntr $ntre e las las sust sustan anci cias as +ue +ue inter nterD Deren eren est'n st'n los los detergentes y las soluciones %'sicas,
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i)etas de auto&'ticas regula%les de =0100 Ul y de =001000 Ul $s)ecto(otó&etro /gua destilada Soluciones est'ndar de )rote*na
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RC$!IMI$NT
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eactivo vo )re)a )re)ara rado do del del sigu siguie ient nte e &odo &odo44 React Reactivo ivo de Bradfo Bradford rd Reacti &e;clar 10 &g de Co&assie Co&assie Blue =<0 con 10 &l de (os(órico al 88 y A- &l de etanol a%soluto, a%soluto, /Eadir ?= hasta 100 &l, iltrar a trav3s de )a)el de Dltro y guardar en la oscuridad, Soluciones estándar de proteínas
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Seroal%6&ina %ovina =0 &gQ&l Seroal%6&ina %ovina 0-< &gQ&l Seroal%6&ina %ovina 0-1 &gQ&l
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Muestras M1 y M2
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5a &uestra M1 de%e tener una concentración de )rote*nas entre 10 V =0 &gQ&l 5a &uestra M= de%e de%e tener una concentraci concentración ón de )rote*nas )rote*nas entre entre 0-1 0-< &gQ&l /Eadir 1 &l del reactivo de Brad(ord a los tu%os est'ndar y &uestras )ro% )ro%le le&a &ass )re) )re)ar arad adas as co&o co&o se indi indica ca en la Ta%la a%la I:, I:, !e !eja jarr a te&)eratura a&%iente y leer /%sor%ancia a << n& (rente al %lanco, $l color es esta%le 1 hora
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M3todo de Biuret
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!eter&inar la concentración de )rote*nas en una &uestra %iológica es una una t3cn t3cnic ica a de ruti rutina na %'si %'sica ca cuan cuando do se a%or a%orda da un es+u es+ue& e&a a de )uriDcación de una )rote*na concreta- cuando se +uiere conocer la actividad es)ec*Dca de una )re)aración en;i&'tica- )ara el diagnóstico de en(er&edades- as* co&o )ara &uchos otros )ro)ósitos, $@isten di(erentes &3todos )ara la cuantiDcación de )rote*nas, Muchos de estos &3todos se %asan en4 a2 la )ro)iedad intr*nseca de las )rote*nas )ara )ara a%so a%sor% r%er er lu; en el ":":- %2 )ara )ara la (or (or&ac &ación ión de deri deriva vado doss +u*&icos- o c2 la ca)acidad +ue tienen las )rote*nas de unir ciertos colorantes .Segal- =00<2,
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5a reacción de %iuret es una reacción coloreada .violeta2 de%ida a la (or&ación de un co&)lejo de Cu en un &edio alcalino en co&)uestos +ue )oseen &'s de un enlace )e)tidico- co&o las )roteinas .Ca&%ell et al- =00G24
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MATERIAL
Mortero !e istilo otas !e ?idro@ido !e Sodio Mechero =A Tu%os !e $nsaye /l%u&ina Cerica%u&ina Cloruro !e Sodio /l 0,
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solu soluci ción ón de 14101410- al no diso disolv lvers erse e %ien %ien se le agre agregar garon on gotas gotas de hidró@ido de sodio hasta llegar a la ho&ogenación deseadadesea da- ta&%i3n o
Se reali;ó reali;ó la solución solución de leche 0,0<&l )ara una solución solución 14=0 y se disolvieron en agua /l &is&o tie&)o se calenta%a agua hasta llegar a una te&)eratura de <00C a <<0C,
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/ continuación- se se)araron =A tu%os de ensaye en dos gru)os de 1= X/ y BY los seis )ri&eros se utili;aron )ara o%tener la curva )atrón y los otros seis )ara o%tener la curva )ro%le&a esto en los dos gru)os- el tu%o 1 se ocu)ó co&o %lanco- al tu%o = 0,1&l- al F 0,=&l- al A 0,A&l- al < 0,8&l- al G 1,0&l de al%u&ina s3rica %ovina .BS/2 con concentración de 10 &gQ&l- res)ectiva&ente,
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$n seguida- los tu%os con 0,=<&l y 8 con 0,<&l de case*na- el con 0,=<&l y el 10 con 0,<&l de so%renadante- el 11 con 0,=< &l y el 1= con 0,<&l de leche diluida- .todo esto o%tenido en la )r'ctica anterior2 a todos los tu%os se les agrego Cloruro de Sodio al 0, con di(erente cantidad de =,&l al =- =,8&l al F- =,G&l al A- =,=&l al < y =&l al G, ara tener tener una solución solución de F&l y reactivo reactivo de Biuret Biuret con la &is&a cantidad cantidad de F &l- as* logra&os una solución solución de G&l- se ho&ogeni;o la solución )or &edio de un agitado ti)o vorte@, .Sola&ente al tu%o 1 se le agregaron F &l de reactivo de %iuret y F &l de NaCl2
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&edic &edició ión n se lava lava%an %an las las celd celdas as al tener tener toda todass las las &edic &edicio ione ness se dis)uso a reali;ar la curva o%tenida, o
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R$S"5T/!S
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5os valores de a%sor%ancia o%tenidos con el es)ectro(otó&etro a
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Tabla 1 Valores de absorbancia de cada una de las muestras ,
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Tu bo !o "
1 = F A < G
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Soluci#n $
F &l NaCl0,1 &l BS/ y =, &l NaCl 0,= &l BS/ y =,8 &l NaCl 0,A &l BS/ y =,G &l NaCl 0,8 &l BS/ y =,= &l NaCl 1 &l BS/ y = &l NaCl 0,=< &l case*na y =,< &l
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%roteín a &'()
0 1 = A 8 10 Z[
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Absor banci a a *+, n' Blanco 0,0FA 0,01 0,1A= 0,= 0,FA< 0,0
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Con los valor valores es de la ta%la ta%la anterioranterior- tu%os tu%os dos a seisseis- se reali reali;ó ;ó la gr'Dca 1- de la curva )atrón de )rote*na- o%tenida )or el &3todo de regr egresió esión n line lineal al de &*ni &*ni&o &oss cuad cuadra rado dos s dond donde e las las a%sc a%scis isas as .@2 .@2 re)resentan la concentración en &g de )rote*na- y las ordenadas .y2 los valores de la a%sor%ancia de cada concentración, .25 .2 .15 .1 Absorbancia a 540 nm
.-5
DEF G .2E H 4I G -
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inter)olación de estos valores en la curva )atrón .Dgura F2 y des)ejado los valores res)ectivos de @ de la ecuación de la recta .y &@ O %2 tal co&o se &uestra a continuación .ta%la =24 o
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o
x=
y −0.0017
0.0344
o
o
Tabla 24 determinación de la cantidad de proteína utilizando
o
la ecuación de recta.
o
o
Tubo No. o o o o o o
7 8 9 10 11 12
o
Absorbancia Absorbancia (y) a 540 nm o o o o o o
0.079 0.198 0.221 0.240 0.044 0.068
o
Proteína (mg) o o o o o o
2.218 5.706 6.375 6.927 1.229 1.927
o
o
To&ando To&ando en cuenta los (actores de disolución- )or cada &ililitro de &uestra se o%tiene la siguiente ta%la4
o
Tabla . Concentración de proteína en las muestras problema
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o
o
3 ml NaCl,
o
0
0
o
o
10
o
Blanc o 0.034
o
0.1 ml BSA y 2.9 ml NaCl
o
1
o
0.2 ml BSA y 2.8 ml NaCl
o
2
o
10
o
0.071
o
0.4 ml BSA y 2.6 ml NaCl
o
4
o
10
o
0.142
o
0.8 ml BSA y 2.2 ml NaCl
o
8
o
10
o
0.277
o
1 ml BSA y 2 ml NaCl
10
o
10
o
0.345
o
0.25 ml caseína y 2.75 ml NaCl
o
o
0.079
o
0.5 ml caseína y 2.5 ml NaCl
o
o
0.198
o
0.221
o
0.240
o
0.044
o
1 o
2 o
3 o
4 o
5 o
o
6 o
7 o
8 o
o
9 o
o
0.25 ml sabrenaan!e y 2.75 ml NaCl 0.5 ml sobrenaan!ey 2.5 ml NaCl
1 o
1
o
0.25 ml lec"e #l$#a y 2.75 ml NaCl 0.5 ml lec"e #l$#a y 2.5 ml NaCl
o
5.70 6
o
6.37 5
o
o o
o
2.21 8
6.92 7 o
o
1.22 9
887. 2 1141 .2 225 138. 54 98.3 2 77.0
0.068
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de a&ino'cidos, 5as )rote*nas ta&%i3n se clasiDcan seg6n su co&)osición- las +ue se (or&an solo de residuos de a&ino'cidos y no tienen otras %io&olecular son RT$]N/S SIM5$S no todas las )rote*nas son solu%les en aguade hecho las )rote*nas insolu%les son esenciales )ara dar so)orte y &antener la integridad estructural de las c3lulas y organis&os, o
o
o
"tili;a&os4 ierce BC/ rotein /ssay Kit .Ther&o ScientiDc2
o
o
o
1re)aración de reactivo BC/ $n 1 tu%o de 10 &l - &e;cla&os Reagent / y Reagent B del ^it- en la siguiente )ro)orción 4 o
o
<0 µl Reagent / Q 1 µl Reagent B o
"tili;a&os )laca de G )ocillos
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o
5o hace&os )or tri)licado )ara tener F lecturas
o
o
$sto se usa )ara generar una &uestra )atrón con µg BS/ en a%scisasy /
o
o
F re)aración de &uestras a cuantiDcar .nor&al&ente )or tri)licado)ero de)ende de la cantidad de &uestra +ue tenga&os y la concentración +ue esti&e&os Xa ojoY4 si cree&os +ue nuestra &uestra & uestra est' &uy concentrada- se )ueden hacer diluciones de uso )ara &edirej, 14=- o 14<- usando %u`er de lisis )ara no ca&%iar la co&)osición+ue )uede a(ectar a las &edidas2,
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o
o
el valor alor &edi &edio o de est estas &edi &edida dass es
=,< =,< O F O F,< F,< F &edidas
o
o
o
o
o
o
o
F µg F µl &uestra
1 µgQµl de )rote*na en nuestro lisado
F µg