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Lecture 12 In- Vitro Translation
Cell Biology (ZOOLOGY 570)
University of Wisconsin-Madison
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Translation Blobel Dobberstein Experiment Expe r i me nt1:I nv i t r ot r ans l at i on ofl i g htc hai n( LC)s ubuni tofant i bodi e s .Li g htc hai n pr o t e i ni sas e c r e t e dpr o t e i n. Expe r i me nt alPr oc e dur e I nt oami c r of uget ubef ol l o wi ngwer eadded:( a. )Ant i bodyl i g htc hai b. )I nv i t r o t r ans l at i on mi x,i . e l y s at e ,l y s at ehad al lt hepr o t e i nsne e de dt odot r ans l at i ( c . ) Radi oac t i vel abe l e d14C ami noac i ds .Fort hee xpe r i me ntant i bodyl i g htc hai nf r om i s ol at e d i nt ac tc e l lwasus e dasac ont r ol . Lys at ef ori nvi t r ot r ans l at i on wasobt ai ned s ubj ec t i ngt hec e l l st o Homo ge ni z ei n or dert obr e akupc e l l sandc ent r i f ugeathi ghs pee ds( 20, ope l l e tal lor gane l l es . Lys at ei st he s uper nat antc ont ai ni ng s ol ubl e pr o t e i ns ,r i bos omes ,t RNA,i ni t i at i on, e l ongat i t er mi nat i onf ac t or s .mRNAwasr emo vedf r om t hel ys at ef ore xper i ment al pur pos e . Radi oi s o t opesc ommonl yus edi nbi ol o gyar e:14C,32P,35S,3H,125I .Advant agesofus i ng r adi oi s o t opes :( a. )Tr ac e abl e( t r ac eamount sofr adi oac t i vemo l e c ul ear epos s i bl et i tt agsmol e c ul e sf ori de nt i fic at i on) b. )Quant i fiabl e( r at eofde c ay) c . )Sens i t i ve ( e as yt ode t e c tandme as ur es mal lamount s ) . Mi c r of uget ubewass ubj ec t edt oSDSPAGE ( Sodi um Dode c y lSul f at ePol yAc r y l ami deGe l El e c t r ophor e s i s )Aut or adi o gr aphyi nor dert os epar at epr o t ei ns . Aut or adi o gr aphy pr o vi des a me an t o vi s ual i z e bi o c hemi c alpr oc es s al l o wi ng an i nve s t i gat ort ode t e r mi net hel oc at i onofr adi oac t i vi t yl abe l edmat er i al swi t hi nac e l l .I n t hi st ec hni que ,t i s s ues ec t i onsc ont ai ni ngr adi oac t i vei s o t opesar ec o ver edwi t h at hi n l ayerofpho t o gr aphi cemul s i on ( fil m) ,whi c hi se xpo s ed r adi at i on emanat i ngf r om r adi oi s o t opeswi t hi nt het i s s ue .Si t esi nt hec e l l sc ont ai ni ngr adi oac t i vi t yar er e ve al ed undert hemi c r os c opes i l vergr ai ni nt heo ver l yi ngemul s i on( fil m) . SDSPAGEAut or adi o gr aphyRe s ul t Ant i bodyl i ghtc hai nmadei nvi t r oappe ar edat25kD whi l el i ghtc hai ni s ol at edf r o mi nt ac t c e l l sappe ar edat21kD. Conc l us i on Di ffe r e nc ei n4kD,l e dt ot hec onc l us i ont hati tmaybeduet os i gnalpe pt i des e que nc eors ome ar t e f ac t . Expe r i me nt2:Whathappe nsi fi nv i t r ot r ans l at i oni sc ar r i e douti nt hepr e s e nc e e nc eof i s ol at e d ER me mbr ane s( mi c r os o me s pr o t e as e ? Pr o t e as ei s an e nz yme t hat de gr ade spr o t e i ns . Expe r i me nt alPr oc e dur e I nt o a mi c r of uge t ube f ol l o wi ng wer e added:( a. )Ant i body l i g htc hai n mRNA ( kno wn s e c r e t i ngpr o t e i n)andGl obi nmRNA ( kno wnc yt os ol i cpr o t e i n) b. )I nv i t r ot r ans l at i onmi x, i . e l y s at e ( c . )Radi oac t i ve l abe l e d 14C ami no ac i ds .For t he e xpe r i me ntpur pos e c yt os o l i cpr o t e i nGl obi n( 14kD)wasus e dasc ont r ol . Mi c r of uget ubewass ubj ec t edt oSDSPAGE ( Sodi um Dode c y lSul f at ePol yAc r y l ami deGe l El e c t r ophor e s i s ) Aut or adi o gr aphy i n or der t os epar at e pr o t ei ns under f our di ffer ent c ondi t i ons . Condi t i on1:Bo t hMi c r os Pr o t e as eabs e nt Condi t i on2:Mi c r os omepr e s e Pr o t e as eabs e nt Condi t i on3:Mi c r os omeabs e Pr o t e as epr e s e nt Condi t i on4:Bo t hMi c r os Pr o t e as epr e s e nt Re s ul t s I nt heabs enc eofMi c r os o Pr o t e as i bodyl i g htc hai nmadei nv i t r oappe ar e dat25 kD Gl obi nappe ar e dat14kD. I nt hepr es enc eofMi c r os abs enc eo fPr o t e as i bodyl i g htc hai nmadei nv i t r o appe ar e dat21kD Gl obi nappe ar e dat14kD. I nt heabs enc eofMi c r os o pr es enc eo fPr o t e as t hi ngappe ar e dbe c aus ebo t ht he pr o t e i nswe r ede gr ade dt hee nz ymepr o t e as e . I nt hepr es enc eofbo t h Mi c r os Pr o t e as yAnt i bodyl i g htc hai n madei nv i t r o appe ar e dat21kD whi l eGl obi npr e s e nc ewasno tde t e c t e dbe c aus eo fi t sde gr adat i ont he e nz ymepr o t e as e . Conc l us i on Di ffe r e nc ei n4kD,l e dt ot hec onc l us i ont hati t st hes i gnalpe pt i des e que nc ec l e ave dt he s i gnalpe pt i das ei nt heRERl ume n. Expe r i me nt3:I sas i gnalpe pt i des uffic i e ntt oge tapr o t e i ni nt ot heRER? Expe r i me nt alPr oc e dur e Gene t i ce ngi neer i ngwasi nvo l ved t omakea c hi me r i cge ne( c hi me r i cpr o t e i n) .Chi mer i c pr o t ei nsbr i ngt wopr o t ei nst o ge t he r . I nt hee xper i mentβLac t amas Gl obi n ge newasus e d asac hi me r i cge newhi l epl ai n Gl obi n ge newasus e d asa c ont r ol .β Lac t amas ei sa s i gnalpe pt i des e c r e t e di n E ol i we i g hi ng3. 5kD tc ons i s t so f28ami noac i ds( 28c odons ) . I nt o a mi c r of uge t ube f ol l o wi ng wer e added:( a. )Pl ai n Gl obi n mRNA ( 14 kD)or β Lac t amas Gl obi n mRNA ( 17. 5 kD) b. )I nv i t r ot r ans l at i on mi x,i . e l y s at e ( c . ) Radi oac t i vel abe l e d14Cami noac i ds .
Lecture 12 In- Vitro Translation
Course: Cell Biology (ZOOLOGY 570)
University: University of Wisconsin-Madison
