Gel Electrophoresis

Post-laboratory Report on Exercise 4.4 Gel Electrophoresis

Vikki Anne R. Cedo CHEM 1!.1 - "# $nd %e&ester $!14-$!1'

Gro(p&ates) *esiree +oy Cerico Ma. ,riselle rnales Mary Ranelle Pasan/

*ate per0or&ed) March 12 $!1' *ate 3nished) March 12 $!1'

Ms. ,orina Vida G. %inad #aboratory 5nstr(ctor

Exercise 4.4

Gel Electrophoresis Po6er0(l electrophoretic techni7(es ha8e been de8eloped to separate &acro&olec(les on the basis o0 their &olec(lar 6ei/ht. 9he &obility o0 a &olec(le in an electric 3eld is in8ersely proportional to the &olec(lar 0riction 6hich is the res(lt o0 its &olec(lar sie and shape: directly proportional to the 8olta/e and char/e o0 the &olec(le. Gel electrophoresis is a techni7(e 6herein &olec(les are separated on the basis o0 their &olec(lar 6ei/ht and char/e. Vario(s /els can be (sed in this techni7(e s(ch as polyacryla&ide and a/arose. Polyacryla&ide /el electrophoresis or PAGE separates polypeptides accordin/ to their &olec(lar 6ei/hts. Proteins that are ne/ati8ely char/ed by the bindin/ o0  the anionic deter/ent sodi(& dodecyl s(l0ate separate 6ithin a &atrix o0  polyacryla&ide /el in an electric 3eld accordin/ to their &olec(lar 6ei/hts.  9able 4.4.1. 5pH;s and direction o0 &i/ration o0 proteins.

Protein

IpH

pH

Pepsin #ysoy&e He&o/lobin Myo/lobin

1.! 11.! . .!

'.! .! .! '.!

Direction of migration Anode Cathode Anode cathode

 

Co-poly&eriation o0 acryla&ide and bis-acryla&ide 0or& the poly&eriation /els. 9he reaction is a 8inyl addition poly&eriation initiated by a 0ree radical/eneratin/ syste& . A&&oni(& pers(l0ate is added to the /el recipe ? /el &ixt(re in order to initiate the /el poly&eriation by acti8atin/ 9EME* or tetra ðyl-ethylenedia&ine. Poly&eriation is initiated by 9EME* and a&&oni(& pers(l0ate. A&&oni(& pers(l0ate yields a pers(l0ate 0ree radical 6hich2 in t(rn2 acti8ates 9EME*. @hen acti8ated2 9EME* acts as an electron carrier to acti8ate the acryla&ide &ono&er into a 0ree radical state. 9his acti8ated &ono&er then reacts 6ith an (nacti8ated &ono&er to be/in the poly&er chain elon/ation <%hi and +acko6ski2 1==>. is-acryla&ide rando&ly crosslinks the elon/atin/ rando& poly&er chains2 6hich then res(lts in closed loops and a co&plex B6ebB poly&er 6ith characteristic porosity that depends on poly&eriation conditions and the &ono&er concentrations. Mi/ration o0 &olec(les2 in /eneral2 are 0aster 6ith hi/her 8olta/e. Mi/ration and 8olta/e ha8e a linear relationship. Acryla&ide concentration2 as 6ell as bis-acryla&ide concentration 2 deter&ines the relati8e sie o0 the pores that 6ill be 0or&ed in the /el. As the total a&o(nt o0 acryla&ide increases2 the pore sie decreases. @e can say that the concentration o0 acryla&ide and the pore sie is in8ersely proportional. ' o0 the cross-linker /i8es the s&allest pore sie 6ith cross-linkin/. Any increase or decrease in the a&o(nt or concentration o0  the cross-linker 6ill res(lt to the increase in pore sie 9he addition o0 beta-

&ercaptoethanol on the /el increases the resol8in/ po6er o0 the /el. eta&ercaptoethanol is a red(cin/ a/ent (sed in breakin/ dis(l3de bonds2 th(s it ens(res that the protein is 0(lly denat(red into its s(b-(nits: beca(se proteins need to be linear in order 0or the PAGE to proceed properly. 5ncreasin/ the te&perat(re to a 8ery hi/h le8el 6ill ca(se the /el to &elt.  9e&perat(re aDects the properties o0 the /el . poly&eriation at !4FC res(lts in t(rbid2 poro(s2 inelastic /els2 and reprod(cibility is dic(lt to achie8e. 9hese properties &ay be d(e to increased hydro/en bondin/ o0 &ono&er at lo6 te&perat(res. Also2 the kinetic ener/y o0 the &olec(les increases 6hen the te&perat(re is increased res(ltin/ to hi/her rate o0 &i/ration.  9able 4.4.$. Molec(lar 6ei/ht or sa&ple and standards and their R0 8al(es.

Protein standards

Lane no.

MW (kDa)

Distanc e travele d by dye 

"

'!! "'! $'!

Distance traveled by sample (cm) $." ".$ ".1

Rf

Log MW

E// alb(&in extract A&&oni(& s(lphate 0raction <!> raction = o0 Gel chro&. Alb(&in isolate raction 1! o0 Gel chro&. Alb(&in isolate raction 11 o0 Gel chro&. Alb(&in isolate

$



!."$ !.4 !.44

$.! $.'4 $.4!

4

=!

"."



!.4

1.='

'

!

".4



!.4=

1.'



'!

".'



!.'

1.!

Calc(lations) R0 I distance tra8elled by sa&ple ? distance tra8elled by trackin/ dye Exa&ple) 0or e// alb(&in extract ".$ ?  I .!"

" $.' $

Log MW of samples and standards

1.' 1 !.' ! !.4" !.44 !.4' !.4 !.4 !.4 !.4= !.' !.'1 Rf val#es

R I -!.=

%lope) -11.1=!4

R$ I !.='

#inear e7(ation) <-11.1=!4x> J .$=

y-intercept) .$= Calc(lations) by interpolation2

log MW $ (%&&.&'!)(Rf val#e)  *.+' Exa&ple) E// 6hite alb(&in) lo/ M@ I <-11.1=!4x> J .$= I $.14$4

MW $ antilog (+.&!+!) $ &,-.- *(e to errors in per0or&in/ the experi&ent2 esti&atin/ the concentrations o0  the sa&ples in the /el2 and /ettin/ the R0 8al(es2 the co&p(ted &olec(lar 6ei/ht is not any6here near the theoretical 8al(e or standard 8al(es. 9he acceptable linear coecient is !.==-1. 5n this experi&ent 6e obtained a ne/ati8e linear coecient2 th(s not achie8in/ the experi&ent;s obKecti8e.

i/(re 4.4.1. Gel electrophoreto/ra& 8is(aliation o0 proteins.

Lati8e PAGE is a ðod co&&only (sed to separate nati8e proteins. 9he conditions o0 the set(p are set so that the proteins that are &i/ratin/ in the /el are kept in their nati8e state. 9he b(Ders (sed in nati8e PAGE pro8eide a nondenat(rin/2 nati8e-like &ilie(: the electrophoresis is per0or&ed at lo6 te&perat(re so the heat can dissipate. L(&ero(s eny&es retain nati8e con0or&ation and eny&atic acti8ities 6hile r(nnin/ in the /el. Lati8e PAGE is also a (se0(l ðod 0or checkin/ the (ni0or&ity o0 an isolated protein. E8en i0 the p(ri3ed protein sa&ple contains only a sin/le type o0 protein2 the sa&ple &i/ht not be (ni0or& s(ch that so&e o0 the &olec(les &i/ht be (n0olded or has (nder/one so&e che&ical &odi3cations. n0oldin/ o0 proteins chan/e the o8erall shape o0 the &olec(le: &ost che&ical &odi3cations chan/e the electric char/e o0 nati8e &olec(les. 50 no side prod(cts or conta&inants are present in the sa&ple2 it is expected to be 8is(alied as a sin/le sharp band2 other6ise2 s&earin/ o0 the band is expected. 5n addition2 nati8e PAGE can also be (sed in the detection o0 co&plex 0or&ation bet6een proteins. 50 a co&plex is present bet6een t6o or &ore proteins 2 the co&plex can be detected as an extra band in the /el. 9his is d(e to the 0act that in nati8e-like conditions2 &any non-co8alent interactions are &aintained and the co&plex &i/rates apparently as a sin/le &olec(le. %*% PAGE or sodi(& dodecyl s(l0ate polyacryla&ide /el electrophoresis is a ðod (sed to separate proteins. Ho6e8er2 (nlike nati8e PAGE2 the proteins in %*% PAGE &i/rate in their denat(red state. 9he &i/ration 8elocity o0 proteins is a 0(nction o0 their sie2 shape and n(&ber o0 electric char/es they carry. As the

8elocity is a co&plex 0(nction o0 these properties2 nati8e PAGE cannot be (sed in esti&atin/ the &olec(lar &ass o0 proteins. 9he traditional nati8e PAGE is si&ilarly (nable to assess 6hether a p(ri3ed protein is co&posed o0 a sin/le s(b(nit or &(ltiple s(b(nits. 9he %*% /el separates indi8id(al polypeptide chains <&ono&eric proteins and s(b(nits o0 &(lti&eric proteins> accordin/ to their sie. %*% is an anionic deter/ent. Proteins treated 6ith %*% at hi/h te&perat(re sho6 radical con0or&ational chan/es. 9his breaks all nati8e non-co8alent inter&olec(lar and intra&olec(lar interactions. M(lti-s(b(nit proteins; s(b(nit str(ct(re disinte/rates and the proteins (n0old. 50 nati8e con0or&ation is stabilied by dis(l3de brid/es2 red(cin/ a/ents are added to open (p connections. %*% &olec(les bind to (n0olded proteins in lar/e excess2 pro8idin/ extra ne/ati8e char/es to the &olec(les. %*% PAGE is a standard ðod 0or assessin/ the ho&o/eneity o0 an isolated protein. 5t is also a rob(st ðod 0or analysis o0 s(pra&olec(lar co&plexes like &(lti-eny&e co&plexes. %*% PAGE separates and denat(res indi8id(al s(b(nits o0  these co&plexes. 9h(s &akin/ the polypeptide chains &i/rate separately in the /el. Vario(s stainin/ proced(res can be (sed to 8is(alie all s(b(nits and relati8e a&o(nts o0 these s(b(nits can be deter&ined or esti&ated. 9his allo6s identi3cation o0 each s(b(nit o0 a co&plex. oth /el 3ltration and electrophoresis are (sed as a separation techni7(e2 their diDerence is that 6hen /el 3ltration is (sed lar/er &olec(les el(tes 3rst and it is non- denat(rin/ 6hile in electrophoresis s&aller &olec(les el(te 3rst and it is denat(rin/. Gel chro&ato/raphy can be (sed 0or desaltin/2 M@ deter&ination and p(ri3cation 6hereas electrophoresis is (sed 0or p(ri3cation2 s(b(nits deter&ination and M@ deter&ination.  9he ad8anta/e o0 (sin/ electrophoresis is that the /els are easy to prepare and that sa&ples can be loaded and r(n si&(ltaneo(sly. Electrophoresis has a 8ery hi/h resol8in/ po6er. 5ts disad8anta/e 6o(ld be that electrophoresis re7(ires the sa&ple to be denat(red and that the e7(ip&ent is expensi8e. 5t also re7(ires electricity and the (se o0 acryla&ide2 a ne(rotoxin: so yo( ha8e to be 8ery care0(l in handlin/ the che&icals and rea/ents 6hen preparin/ the /el. Gel 3ltration chro&ato/raphy2 the &o8e&ent o0 the &olec(les is only based on the /ra8itational p(ll: beca(se yo( 6ill K(st 6ait 0or the &olec(les to el(te o(t o0  the /el. 5t only re7(ires si&ple e7(ip&ent in order to be per0or&ed and the protein sa&ples do not ha8e to be denat(red in the process2 keepin/ intact their nati8e con0or&ation and biolo/ical acti8ity. 9he disad8anta/es o0 the techni7(e incl(de) it is ti&e cons(&in/ and &ore eDort is needed beca(se o0 the preparation o0 the sie8in/ col(&n2 collection o0 0ractions is done one-by-one and this takes a lot o0  ti&e2 &(st be &onitored 0ro& ti&e to ti&e and the resol8in/ po6er is not eDecti8e eno(/h to separate 8ery sli/ht diDerences in M@.

Literat#re ited ettelhei&2 . <$!!>. Introduction to General, Organic and Biochemistry. rooks?Cole by 9ho&son #earnin/. Chen2 . and Chra&bach2 A.2 <1==>. Esti&ation o0 poly&eriation eciency in the 0or&ation o0 polyacryla&ide /el2 (sin/ contin(o(s optical scannin/ d(rin/ poly&eriation2 + ioche&istry and iophysics Methods 12 1!'-11 Chra&bach2 A.2 <1='>. 9he Practice o0 N(antitati8e Gel Electrophoresis2 VCH2 *eer3eld each. %hi2 N. and +acko6ski2 G.2 <1==>. ne di&ensional polyacryla&ide /el electrophoresis2 pp 1-'$ in Harnes * Gel Electrophoresis o0 Proteins) A Practical Approach2 "rd edition2 x0ord ni8ersity Press2 x0ord. @an/2 L. %. . Chemical & Biomolecular Engineering Ofcial Site.  Retrie8ed March $2 $!1'2 0ro& Maryland ni8ersity *epart&ent o0 Che&ical O io&olec(lar En/ineerin/) http)??666.en/.(&d.ed(?ns6?ench4'?labc.ht&