Tehnici moderne +

7/25/2019 Tehnici moderne +

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TEHNICI MODERNE N

BIOLOGIA CELULAR


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STUDIUL PROCESELORCELULARE

2 aspecte:2 aspecte:

Tehnici si et!"e a"ec#ate "eTehnici si et!"e a"ec#ate "e$%$%&&%i%e a 'en!ene(!% ce($(a%e%i%e a 'en!ene(!% ce($(a%e

M!"e(a%ea e)pe%ienta(M!"e(a%ea e)pe%ienta(&&aa

'en!ene(!% ce($(a%e'en!ene(!% ce($(a%e


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MODALIT*I DE STUDIU

+,+, in vivoin vivo

2,2, in situin situ

-,-, in vitroin vitro


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MODALIT*I DE STUDIU

in vivoin vivo . can" ce($(e(e sa$ !%/ane(e a(e ca%$i. can" ce($(e(e sa$ !%/ane(e a(e ca%$ice($(e "!%i sa (e st$"ie s$nt a0!%"ate ince($(e "!%i sa (e st$"ie s$nt a0!%"ate in!%/anis$( intact!%/anis$( intact,,

A#anta1e: %e$(tate(e !03in$te s$nt e"i'icat!a%eA#anta1e: %e$(tate(e !03in$te s$nt e"i'icat!a%epent%$ p%!ces$( st$"iat (a ni#e( "e !%/anispent%$ p%!ces$( st$"iat (a ni#e( "e !%/anis

Dea#anta1e: %Dea#anta1e: %e$(tate(e e)pe%iente(!% s$nte$(tate(e e)pe%iente(!% s$nt"i'ici( "e inte%p%etat"i'ici( "e inte%p%etat "at!%it&"at!%it&in'($enin'($en33e(e(!%!%ce(!%(a(te c!p!nente a(e !%/anis$($ice(!%(a(te c!p!nente a(e !%/anis$($i


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MODALIT*I DE STUDIU

in situin situ4 can" !%/an$( "e inte%es este4 can" !%/an$( "e inte%es esteentin$t (a (!c$( sa$ in !%/anis "a%entin$t (a (!c$( sa$ in !%/anis "a%s$nt c!nt%!(ate $ne(e in'($ente c$s$nt c!nt%!(ate $ne(e in'($ente c$

s$nt ce(e ne%#!ase sa$ en"!c%ines$nt ce(e ne%#!ase sa$ en"!c%ine

A#anta1e: se p!t st$"ia #a%ia3ii(eA#anta1e: se p!t st$"ia #a%ia3ii(ep%!ces$($i 5n c!n"i3ii c!nt%!(atep%!ces$($i 5n c!n"i3ii c!nt%!(ate

Dea#anta1e: n$ se p!ate i"enti'icaDea#anta1e: n$ se p!ate i"enti'ica%e$(tat$( c$ ce( 5n #i#!%e$(tat$( c$ ce( 5n #i#!


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MODALIT*I DE STUDIU

in vitroin vitro4 can" !%/an$( 64 can" !%/an$( 6 33es$t$( sa$es$t$( sa$ce($(e(e s$nt i!(ate "in !%/anisce($(e(e s$nt i!(ate "in !%/anis

A#anta1e:A#anta1e: se pe%ite $n c!nt%!(se pe%ite $n c!nt%!(%i/$%!s a( e)pe%ient$($i,%i/$%!s a( e)pe%ient$($i,

Dea#anta1e: n$ se p!ate /a%antaDea#anta1e: n$ se p!ate /a%anta!03ine%ea ace(!%a7i %e$(tate in #i#!,!03ine%ea ace(!%a7i %e$(tate in #i#!,


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Tehnici in #it%!

Baia "e !%/anBaia "e !%/an

C$(t$%a "e ce($(eC$(t$%a "e ce($(e


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Baia "e !%/an

.. pe%ite enpe%ite en33ine%ea #ia0i(itine%ea #ia0i(it&3&3ii $n!% '%a/enteii $n!% '%a/ente"e !%/an sa$"e !%/an sa$ 33es$t$%i,es$t$%i,

..#a%iate 5#a%iate 5n '$ncn '$nc33ie "e !%/an$( s$p$sie "e !%/an$( s$p$se)pe%ient$($ie)pe%ient$($i

..asi/$%asi/$%&&! tepe%at$%a c!nstant! tepe%at$%a c!nstant&&6 !)i/ena%ea6 !)i/ena%eaate%ia($($i 0i!(!/ic 6 p!si0i(itatea c!nt%!(ate%ia($($i 0i!(!/ic 6 p!si0i(itatea c!nt%!(&&%ii%ii

c!p!ic!p!i33iei e"i$($i "in 0aieiei e"i$($i "in 0aie 77i c$(e/e%ea "ei c$(e/e%ea "e"ate '$nc"ate '$nc33i!na(ei!na(e 77i ei e77anti!aneanti!ane


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C$(t$%i "e ce($(e . Etape DigestiaDigestiaenzimaticaenzimatica a tes$t$($i c$ !0tine%eaa tes$t$($i c$ !0tine%ea

$nei s$spensii "e ce($(e$nei s$spensii "e ce($(e IzolareaIzolarea$nei p!p$nei p!p$$(a(a33ii ce($(a%e p$%eii ce($(a%e p$%e

IInsnsmmnnareaarea55n '(ac!ane "e c$(t$%n '(ac!ane "e c$(t$%&&sa$ p(sa$ p(&&ciciPet%i !0Pet%i !033inin88n"$.se ast'e( ! c$(t$%n"$.se ast'e( ! c$(t$%&&p%ia%p%ia%&& PasareaPasarea --%es$spen"a%ea ce($(e(!% "in c$(t$%a%es$spen"a%ea ce($(e(!% "in c$(t$%a

p%ia%a $%atp%ia%a $%at

&&

"e "e %e"e "e %e

55

nsns

&&

88

nn

33a%eaa%ea

55

nn

'(ac!ane "e c$(t$%'(ac!ane "e c$(t$%&&sa$ p(sa$ p(&&ci Pet%i !0ci Pet%i !033inin88n"$.n"$.se c$(t$%i sec$n"a%se c$(t$%i sec$n"a%ee


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C$(t$%i ce($(a%e . E#!($3ie

+,+, Faza de lagFaza de lagsa$ "e in"$csa$ "e in"$c33ieie .. ce($(e(ece($(e(e5757ii%e'ac s$p%a'a%e'ac s$p%a'a33aa 77i se ac!!"eai se ac!!"ea&&c$ e"i$(c$ e"i$("e c$(t$%"e c$(t$%&&

2,2, 99aza exponenaza exponenialial.. ce($(e(e se "i#i" %api"6ce($(e(e se "i#i" %api"6e)p!nene)p!nen33ia(ia(-,-, Faza staFaza staionarionar.. nn$&$&%%$($("e ce($(e %"e ce($(e %&&88nene

c!nstant, In aceastc!nstant, In aceast&&'a'a&&se e'ect$ease e'ect$ea&&

pasa%eapasa%ea,, Faza de declinFaza de declin.. scsc&&"e%ea n%, "e ce($(e"e%ea n%, "e ce($(e

"at!%it"at!%it&&5500&&t%t%88ni%iini%ii 77i !%i !%33ii ,ii ,


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Me"ii "e c$(t$%& .C!p!nente

+,+, Ai!aciiAi!acii 7i #7i #itaineitaine2,2, SS&&%$%i 4 necesa%e en%$%i 4 necesa%e en33ine%ii echi(i0%$($iine%ii echi(i0%$($i

i!nici!nic 77i pH e"i$($i "e c$(t$%i pH e"i$($i "e c$(t$%&&

-,-, G($c!a pent%$ necesa%$( ene%/eticG($c!a pent%$ necesa%$( ene%/etic,, Se% 'eta(Se% 'eta(

;,;, Anti0i!ticeAnti0i!tice


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+,+, MMorfologia si morfometriaorfologia si morfometria2,2, Citometria in fluxCitometria in flux3.3. Fractionarea celularaFractionarea celulara4.4. Citochimia si citoenzimologiaCitochimia si citoenzimologia

5.5. ImunocitochimiaImunocitochimia 6.6. Fluorescenta si imunofluorescentaFluorescenta si imunofluorescenta

.. !icrofiziologia si microin"ectia!icrofiziologia si microin"ectia #.#. $adioautografia$adioautografia%.%. !etode &iochimice!etode &iochimice'(.'(. )ehnici si metode de &iologie moleculara)ehnici si metode de &iologie moleculara


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!*$F*!+)$I,

. $ti(iea& $n$ti(iea& $n analizor de imagineanalizor de imagine,,

. se sta0i(e7tese sta0i(e7te c%ite%i$( "$pa ca%e se #ac%ite%i$( "$pa ca%e se #ae'ect$a ana(iae'ect$a ana(ia6 p%ec$ 7i (iite(e a)i&6 p%ec$ 7i (iite(e a)i&

7i ini& a(e acest$ia7i ini& a(e acest$ia. Re$(tat:Re$(tat: "i'e%it"i'e%iteep!p$(ap!p$(a33ii ce($(a%e,ii ce($(a%e,

Re$(tat$( ana(iei cantitati#e este a'isat s$0Re$(tat$( ana(iei cantitati#e este a'isat s$0'!%a $nei hist!/%ae,'!%a $nei hist!/%ae,


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Citometria -n flux

.. ppee%ite ana(ia%ite ana(ia 77i sepa%a%ea ce($(e(!% pei sepa%a%ea ce($(e(!% pe0aa $n!% ca%acte%e !%'!(!/ice0aa $n!% ca%acte%e !%'!(!/ice 77ii0i!chiice0i!chiice

O'e%O'e%&&in'!%ain'!%a33ii as$p%a :ii as$p%a :+, +, &&%iii %e(ati#e a ce($(e(!%%iii %e(ati#e a ce($(e(!%

2, /%an$(a%it2, /%an$(a%it&3&3ii inte%ne =ne!!/enitateaii inte%ne =ne!!/enitateainte%ninte%n&&cit!p(asaticcit!p(asatic&&> %e(ati#> %e(ati#&&

-, intensitatea %e(ati#-, intensitatea %e(ati#&&a '($!%escena '($!%escen33eiei,,


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Citometria -n flux utilitate

+,+, &&s$%a%ea ca%acte%istici(!% 'iice a(es$%a%ea ca%acte%istici(!% 'iice a(ece($(eice($(ei2, i"enti'ica%ea si sepa%a%ea p!p$(a2, i"enti'ica%ea si sepa%a%ea p!p$(a33ii(!%ii(!%

"e ce($(e san/$ine = /%an$(!cite6"e ce($(e san/$ine = /%an$(!cite6!n!cite 6 (i'!cite T sa$ B>!n!cite 6 (i'!cite T sa$ B>-, -, &&s$%a%ea cantits$%a%ea cantit&3&3ii "e ADii "e ADNN"in ce($("in ce($(&&

, , &&s$%a%ea cantits$%a%ea cantit&3&3ii int%ace($(a%e "eii int%ace($(a%e "eCaCa;, as$%a%ea pH.$($i int%ace($(a%;, as$%a%ea pH.$($i int%ace($(a%


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Citometria in flux


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Fracionarea celular

.. tehnictehnic&&ce pe%ite i!(a%eace pe%ite i!(a%ea"i'e%ite(!% e(eente $(t%ast%$ct$%a(e"i'e%ite(!% e(eente $(t%ast%$ct$%a(esi 0i!chiice a(e ce($(e(!%si 0i!chiice a(e ce($(e(!%


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Fracionarea celular

+tape *mogenizarea*mogenizarea4"ist%$/e%ea inte/%it4"ist%$/e%ea inte/%it&3&3iiiitis$(a%etis$(a%e 77i ap!i ce($(a%e c$ !0i ap!i ce($(a%e c$ !033ine%eaine%ea$nei s$spensii "e !%/anite$nei s$spensii "e !%/anite 77iie0%ane #eic$(atee0%ane #eic$(ate55n e"i$( "en e"i$( "e!!/enia%e!!/enia%e

/epararea/epararea p%in cent%i'$/a%ep%in cent%i'$/a%e 77i? sa$i? sa$$(t%acent%i'$/a%e$(t%acent%i'$/a%e


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*!*0+1I2,$+,

a, p%ina, p%in soc osmoticsoc osmotic.. Di'e%enDi'e%en33a "e !s!(a%itatea "e !s!(a%itate55nt%e e"i$( i,c, si ce( e,c, "$ce (a $'(a%eant%e e"i$( i,c, si ce( e,c, "$ce (a $'(a%eace($(e(!% pana (a %$pe%ea e0%ane(!%,ce($(e(!% pana (a %$pe%ea e0%ane(!%,

0, p%in0, p%in ultrasonicareultrasonicare4 p%es$p$ne %$pe%ea4 p%es$p$ne %$pe%eae0%ane(!% s$0 ace0%ane(!% s$0 ac33i$nea $n"e(!% $(t%as!nicei$nea $n"e(!% $(t%as!nice

c,c, cu omogenizatoarecu omogenizatoarese %ea(ieaa s$0se %ea(ieaa s$0

acac33i$nea '!%i$nea '!%33e(!% "e '%eca%ee(!% "e '%eca%e 77i t!%si$ne ca%ei t!%si$ne ca%ec!nt%i0$ie (a %$pe%ea e0%ane(!%,c!nt%i0$ie (a %$pe%ea e0%ane(!%,


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/+,$,$+,

diferendiferenialial4 can" !%/anite(e se4 can" !%/anite(e se"ep$n (a 0aa t$0$($i 6"ep$n (a 0aa t$0$($i 655n !%"inean !%"inea"ensit"ensit&3&3ii (!% p%in cent%i'$/a%i %epetateii (!% p%in cent%i'$/a%i %epetate(a '!%(a '!%33e "in cee "in ce55n ce ai a%in ce ai a%i

--n gradient de densitaten gradient de densitate4p%int%.!4p%int%.!sin/$%sin/$%&&cent%i'$/a%e (a '!%cent%i'$/a%e (a '!%33e s$'iciente s$'icient"e %i"icate se !0"e %i"icate se !033in !%/anite(e ca '%acin !%/anite(e ca '%ac33iiiisepa%ate ,sepa%ate ,


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N U C L E I

U l t ra s o n i c a re

M ito c o n d r i i

L i z o z o m i

M e m b ra n ec e lu la reG o l g i

m i c ro s o m i

R ib o s o m i re t ic u l e n d o p la s m i c

P u r i f c a re in

g ra d ie n td e d e n s ita t e

S e p a ra re a c o n s t i tu e n t i lo rc e lu la r i p r i n v i t e z e d e c e n t ri ! u g a re c re s c a n d e


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,)*$,I*0$,FI,

..tehnica "e in#esti/a%e ce($(a%tehnica "e in#esti/a%e ce($(a%&&ce pe%itece pe%itei"enti'ica%ea !%'!(!/ici"enti'ica%ea !%'!(!/ic&&77i $(t%ast%$ct$%a(i $(t%ast%$ct$%a(&&aa$n!% c!p$$n!% c!p$77i %a"i!acti#i,i %a"i!acti#i,

t%as!%i %a"i!acti#it%as!%i %a"i!acti#i ::. ain!acii6 /($ci"e6 n$c(e!ti"eain!acii6 /($ci"e6 n$c(e!ti"e

. '!s'at sa$ s$('at %a"i!acti#'!s'at sa$ s$('at %a"i!acti#

. (i/ani %a"i!acti#i speci'ici(i/ani %a"i!acti#i speci'ici

Marcarea unorMarcarea unor

componentecomponente

celularecelulare

receptori saureceptori sauanticorpi radioactivianticorpi radioactivi


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!+)*+ I*C7I!IC+

+8+C)$*F*$+2,+8+C)$*F*$+2,

I!1*+8+C)$*F*$+2,I!1*+8+C)$*F*$+2,

)$,1/F+$8 +8+C)$*F*$+)IC)$,1/F+$8 +8+C)$*F*$+)IC


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+8+C)$*F*$+2,

PRINCIPIU:PRINCIPIU: pa%tic$(e(epa%tic$(e(e55ncnc&&%cate%catee(ect%ic i/%eae(ect%ic i/%ea&&55nt%.$n e"i$nt%.$n e"i$##88sc!s 6 s$0 acsc!s 6 s$0 ac33i$nea $n$i ci$nea $n$i c88ppe(ect%ic,e(ect%ic,

Ap(ica3ii p%actice:Ap(ica3ii p%actice:st$"i$( p%!teine(!%st$"i$( p%!teine(!% 77i ai aacii(!% n$c(eici,acii(!% n$c(eici,


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+8+C)$*F*$+2,

$*)+I1+8*$a,a, unidimensionalunidimensional6 can" i/%a%ea se6 can" i/%a%ea se

%ea(iea%ea(iea&&55n /e(n /e(55nt%.! sin/nt%.! sin/$$%%&&"i%ec"i%ec33ieie

0,0, &idimensional&idimensional6 c6 c88n" i/%a%ea sen" i/%a%ea se%ea(iea%ea(iea&&s$ccesi#s$ccesi#55n "!$n "!$&&"i%ec"i%ec33iiii

Ap(ica3ii p%actice 4 e(ect%!'!%ea $ni"iensi!na(&Ap(ica3ii p%actice 4 e(ect%!'!%ea $ni"iensi!na(& SDS 4 PAGESDS 4 PAGE 9OCALI@AREA I@OELECTRICA9OCALI@AREA I@OELECTRICA


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ELECTRO9ORE@A SDS.

PAGE..%ea(iea%ea(iea&&sepa%a%ea p%!teine(!% "int%.$nsepa%a%ea p%!teine(!% "int%.$naestec pe 0aa /a0a%it$($i !(ec$(a%aestec pe 0aa /a0a%it$($i !(ec$(a%"i%ect p%!p!%"i%ect p%!p!%33i!na( c$ asa !(ec$(a%i!na( c$ asa !(ec$(a%&&,,

P%incipi$P%incipi$:: a"s!%0a"s!%033iiaa"!"eci(s$('at"!"eci(s$('at$($i$($i"e"es!"i$ $nita% pe p%!teines!"i$ $nita% pe p%!teine66%ea(i%ea(i88n" !n" !"ensitate "e sa%cin"ensitate "e sa%cin&&ne/ati#ne/ati#&&$ni'!%$ni'!%&&,,

ELECTRO9ORE@A SDS


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ELECTRO9ORE@A SDS.PAGE


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Focalizarea izoelectrica

. sepa%a%ea p%!teine(!% "int%.$n aestecsepa%a%ea p%!teine(!% "int%.$n aestecse 'ace pe 0aa p$nct$($i (!% i!e(ect%ic,se 'ace pe 0aa p$nct$($i (!% i!e(ect%ic,

P%incipi$: i/%a%ea pP%incipi$: i/%a%ea p%!t%!teeine(ine(!%!%55n '$ncn '$nc33ieie"e p$nct$( i!e(ect%ic6"e p$nct$( i!e(ect%ic6 p8n& 5np8n& 5n !na c$!na c$pH.$( i"entic c$ ce( a( p$nct$($i (!%pH.$( i"entic c$ ce( a( p$nct$($i (!%

i!e(ect%ici!e(ect%ic6 $n"e6 $n"e"e#in ne$t%e !p%in"$."e#in ne$t%e !p%in"$.77iiisca%ea,isca%ea,


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Focalizarea izoelectrica

" e n z i p ro t e i c e la p u n c t u l i z o e le c t r ic


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+lectroforeza

&idimensionala..i/%a%ea p%!teine(!%i/%a%ea p%!teine(!%55n "!$n "!$&&"i%ec"i%ec33ii :ii :+, in p%ia "i%ec+, in p%ia "i%ec33ieie55n '$ncn '$nc33ie "e p$nct$( (!%ie "e p$nct$( (!%

i!e(ect%ici!e(ect%ic ..9OCALI@ARE I@OELECTRICA9OCALI@ARE I@OELECTRICA

2, a "!$a "i%ec2, a "!$a "i%ec33ieie ..SDS 4 PAGE 6SDS 4 PAGE 655n '$ncn '$nc33ieie"e /%e$tatea !(ec$(a%"e /%e$tatea !(ec$(a%&&a p%!teine(!%,a p%!teine(!%,

Re$(tat$( se p%einta s$0 '!%a $neiRe$(tat$( se p%einta s$0 '!%a $nei hh&&%%33i c$i c$p%!teine(e sepa%atep%!teine(e sepa%ate55n p(an$( /e($($i,n p(an$( /e($($i,

+l t f


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+lectroforeza&idimensionala

E le c tro ! o r e z a l a p u n c t iz o e le c t r ic

S D S -


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I!1*+8+C)$*F*$+2,

.. ! #a%iant! #a%iant&&a e(ect%!'!%eei ce c!0ina e(ect%!'!%eei ce c!0in&&i/%a%eai/%a%ea55n cn c88p e(ect%ic a p%!teine(!% c$p e(ect%ic a p%!teine(!% c$%ea(ia%ea $nei %eactii i$ne%ea(ia%ea $nei %eactii i$ne "e tip A/.Ac"e tip A/.Ac

Reactia i$naReactia i$na::.."$pa te%ina%ea i/%a%ii e(ect%!'!%etice6"$pa te%ina%ea i/%a%ii e(ect%!'!%etice6

.

.c!nc!itentc!nc!itent

==i$n!e(ect%!'!%eai$n!e(ect%!'!%ea

55

n %achetn %achet

&> .&> .

ap%ecie%e cantitati#ap%ecie%e cantitati#&&a A/a A/ ==s$p%a's$p%a'a3aa3a"esc%is"esc%is&&"e (inia "e p%ecipita%e c$ (inia "e sta%t"e (inia "e p%ecipita%e c$ (inia "e sta%t>>,,


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I!1*+8+C)$*F*$+2,

)$,1/F+$8


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)$,1/F+$8+8+C)$*F*$+)IC

..et!"et!"&&"e e)t%a/e%e a p%!teine(!%"e e)t%a/e%e a p%!teine(!%sepa%ate e(ect%!'!%etic "int%.$n /e( 6 c$sepa%ate e(ect%!'!%etic "int%.$n /e( 6 c$a"s!%0i%ea (!% pe niste ha"s!%0i%ea (!% pe niste h88%tii c$ p%!p%iet%tii c$ p%!p%iet&3&3ii

specia(e,specia(e,

P%!teine(eP%!teine(e s$'e%&s$'e%& %eac%eac33ii "e %ec$n!aii "e %ec$n!a77te%ete%e

speci'icspeci'ic&&p%in inte%e"i$( $n!% inte%acp%in inte%e"i$( $n!% inte%ac33i$nii$ni"e tip %ecept!% 4 (i/an" sa$ A/ 4 A"e tip %ecept!% 4 (i/an" sa$ A/ 4 Acc,,


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B(!t$%i(e

. Met!"& "e t%ans'e% a $n!% p%!teine6Met!"& "e t%ans'e% a $n!% p%!teine6sec#ente ADN sa$ ARN6 "int%.$n /e(sec#ente ADN sa$ ARN6 "int%.$n /e(

e(ect%!'!%etic pe $n s$p!%t ca%e pe%itee(ect%!'!%etic pe $n s$p!%t ca%e pe%ite#i$a(ia%ea (!% $(te%i!a%i$a(ia%ea (!% $(te%i!a%&

. Uti(iate pent%$ i"enti'ica%ea p%een3eiUti(iate pent%$ i"enti'ica%ea p%een3ei

$n!% p%!teine? sec#en3e ADN 5nt%.$n$n!% p%!teine? sec#en3e ADN 5nt%.$n(iat ce($(a%(iat ce($(a%


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S!$the%n B(!t

. Uti(iat pent%$ i"enti'ica%ea $nei an$iteUti(iat pent%$ i"enti'ica%ea $nei an$itesec#en3e ADN6 p%in '!(!si%ea $nei p%!0esec#en3e ADN6 p%in '!(!si%ea $nei p%!0e

ce c!n3ine sec#en3a c!p(eenta%&ce c!n3ine sec#en3a c!p(eenta%&ce(ei c&$tate,ce(ei c&$tate,. P%!0a "e hi0%i"ia%e este a%cat&P%!0a "e hi0%i"ia%e este a%cat&

'($!%escent sa$ %a"i!/%a'ic6 pe%i38n"'($!%escent sa$ %a"i!/%a'ic6 pe%i38n""epista%ea $(te%i!a%& a p%epa%ate(!% ca%e"epista%ea $(te%i!a%& a p%epa%ate(!% ca%ec!n3in sec#en3a c&$tat&c!n3in sec#en3a c&$tat&


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N!%then B(!t

. Detecta%ea an$it!% sec#ente "e ARNDetecta%ea an$it!% sec#ente "e ARN

. Sii(a%& ca p%!ce"$%& S!$the%n B(!t 4Sii(a%& ca p%!ce"$%& S!$the%n B(!t 4

$($i$($i. 9$%niea& in'!%a3ii (e/ate "e9$%niea& in'!%a3ii (e/ate "e

e)p%ia%ea an$it!% /ene 5n an$itee)p%ia%ea an$it!% /ene 5n an$ite

p!p$(a3ii ce($(a%ep!p$(a3ii ce($(a%e


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este%n B(!t.

i"enti'ica%ea $nei an$itei"enti'ica%ea $nei an$itep%!teine 5nt%.$n !!/eniatp%!teine 5nt%.$n !!/eniattis$(a%tis$(a%.

P%!teine(e "in p%epa%at s$ntP%!teine(e "in p%epa%at s$ntsepa%ate p%in e(ect%!'!%e& 5nsepa%ate p%in e(ect%!'!%e& 5n/e(6 ap!i t%ans'e%ate pe !/e(6 ap!i t%ans'e%ate pe !e0%an& s$p!%t $n"e6 p%ine0%an& s$p!%t $n"e6 p%ina"&$/a%ea Ac speci'ic6 se #aa"&$/a%ea Ac speci'ic6 se #ai"enti'ica p%een3a?a0sen3ai"enti'ica p%een3a?a0sen3ap%!teinei sa$ cantitatea ei 5np%!teinei sa$ cantitatea ei 5n

p%!0a "at&,p%!0a "at&,
http://en.wikipedia.org/wiki/Image:Western_blot.jpg


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este%n B(!t

Uti(i&%i:Uti(i&%i: Cea ai '!(!sit& et!"& pent%$Cea ai '!(!sit& et!"& pent%$

"ete%ina%e cantitati#& "e p%!teine"ete%ina%e cantitati#& "e p%!teine O'e%& in'!%a3ii (e/ate "e !"i'ica%eaO'e%& in'!%a3ii (e/ate "e !"i'ica%ea

cantitati#& a $nei p%!teine 5n '$nc3ie "ecantitati#& a $nei p%!teine 5n '$nc3ie "e

tip$( ce($(a% sa$ t%ataente(e ap(icatetip$( ce($(a% sa$ t%ataente(e ap(icate$nei p!p$(a3ii ce($(a%e$nei p!p$(a3ii ce($(a%e

PCR =POLMERASE CHAIN


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PCR =POLMERASE CHAINREACTION>

. 9!(!sit& pent%$ !03ine%ea $n$i n$&% a%e. 9!(!sit& pent%$ !03ine%ea $n$i n$&% a%e"e c!pii a $nei an$ite sec#en3e "e ADN"e c!pii a $nei an$ite sec#en3e "e ADN

EtapeEtape

+, "enat$%a%ea ADN c$ sepa%a%ea catene(!%+, "enat$%a%ea ADN c$ sepa%a%ea catene(!%c!p(eenta%ec!p(eenta%e

2, a"a$/a%ea p%ie%i(!% =sec#en3e ADN2, a"a$/a%ea p%ie%i(!% =sec#en3e ADN

c!p(eenta%e sec#en3ei.3int&>c!p(eenta%e sec#en3ei.3int&>-, s$0 ac3i$nea Ta.p!(ie%aei se-, s$0 ac3i$nea Ta.p!(ie%aei sesintetiea& n!i sec#en3e "e ADN "csintetiea& n!i sec#en3e "e ADN "c


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RT 4 PCR =REERSETRANSCRIPTASE PCR>

. Met!"& '!a%te sensi0i(& "e "etecta%e 7iMet!"& '!a%te sensi0i(& "e "etecta%e 7ic$anti'ica%e a ARNc$anti'ica%e a ARN

. Ini3ia( "in ARN.$( 3int& se sintetiea&Ini3ia( "in ARN.$( 3int& se sintetiea&sec#en3a cADN6 ap!i $%ea& etape(e PCRsec#en3a cADN6 ap!i $%ea& etape(e PCR

. Uti(iat& 5n "ia/n!stic$( $n!% 0!(i /enetice6Uti(iat& 5n "ia/n!stic$( $n!% 0!(i /enetice6c$anti'ica%ea e)p%esiei "i#e%se(!% /ene p%inc$anti'ica%ea e)p%esiei "i#e%se(!% /ene p%in

"ete%ina%ea cantit&3ii "e ARN"ete%ina%ea cantit&3ii "e ARN


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Ania(e t%ans/enice

. Ania(e "e (a0!%at!% c&%!%a (i se inse%&Ania(e "e (a0!%at!% c&%!%a (i se inse%&/ene $tante pent%$ a st$"ia e'ect$( (!%/ene $tante pent%$ a st$"ia e'ect$( (!%as$p%a !%/anis$($i /a"&,as$p%a !%/anis$($i /a"&,

. Me"i$ "e st$"i$ pent%$ e#!($3ia $n!% 0!(i c$Me"i$ "e st$"i$ pent%$ e#!($3ia $n!% 0!(i c$"ete%inis /enic 7i a !p3i$ni(!%"ete%inis /enic 7i a !p3i$ni(!%te%ape$ticete%ape$tice


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Ania(e "e (a0!%at!% t%ans/enice


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Ania(e Fn!cF!$t

. n(!c$i%ea /enei "e inte%es c$ ! /en&n(!c$i%ea /enei "e inte%es c$ ! /en&inacti#& sa$ ! a(e(& $tat&inacti#& sa$ ! a(e(& $tat&

. Ania($( p$%t&t!% n$ #a e)p%iaAnia($( p$%t&t!% n$ #a e)p%iaaceast& /en&6 p$t8n"$.se st$"ia e'ect$(aceast& /en&6 p$t8n"$.se st$"ia e'ect$(a0sen3ei ei in !%/anis,a0sen3ei ei in !%/anis,


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