Purifikasi Protein

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    Pedoman pemurnianprotein :1. Menentukan tujuan Kemurnian, aktivitas, kuantitas yang diperlukan untuk

    produk akhir untuk menghindari kelebihan ataukekurangan metode yang digunakan.

    2. Menentukan sifat (properties) dari protein target

    dan batas kritis pengotor, untuk menyederhanakanteknik seleksi dan optimasi.

    3. Menentukan metode analisis yang tepat, untukmempercepat deteksi aktivitas/recovery protein agar

    pekerjaan efsien.4. Meminimalkan penanganan sampel pada setiap

    tahap

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    $lasan melakukan pemurnian

    protein%ntuk menentukan &%'* protein

    %ntuk menentukan +%K+% protein

    %ntuk penggunaan pemurnian pemurnianproduct -*'+*0*$+1 dalam alur reaksi/processing

    %ntuk memproduksi produk 232*$4

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    +$+* P%'*$'

    P3+*'• %ntuk penggunaan apa protein diproduksi5

    • 0ari organisme apa, pada bagian mana 5

    • $pakah protein intraseluler atau ekstraseluler5

    •  6ika intraseluler terletak di bagianmana pada sel5

    • 7agaimana menanganinya5

    • 7erapa kemurnian yang dibutuhkan yangberhubungan dengan sumber protein dan produkakhir5

    •  6ika dilakukan cale1up (skala besar) teknik apa yangdiperlukan, berkaitan efsiensi 8 sumber danketersediaan peralatan.

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     +iga +ahap trategi

    Purifkasi!ujuan utama untuk memurniakan proteinadalah untuk mengisolasi, mendapatkankonsentrasi dan stabiltas protein sesuai dengantujuan pemurnian target.

    "ase purikasi intermediate, bertujuan untukmembuang sebagian pengotor, misalnya proteinlain, asam nukleat, endoto9in, dan virus.

    "ase terakhir bertujuan untuk mendapatkan

    kemurnian tinggi, menyingkirkan semua pengotoratau senyaa yang berhubungan dengan proteintarget.

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    Kombinasi eleksi dan +ahap Purifkasi yang3ptimum :%ntuk menjamin perkembanganmetode yang cepat, aktu yang

    singkat untuk mendapatkanproduk yang murni, dankeuntungan ekonomi.

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    Preparasi#$%& (+ujuan akhir) : kemurnian dan kuantitas tinggi,

    maintanace (pemeliharaan) aktivitas biologi, sehinggamemenuhi syarat ekonomi dan aktu.

    !entukan kemurnian yang dibutuhkan oleh produkakhir

    2ontoh kemurnian yang dibutuhkan :Kemurnian angat +inggi ;

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    *dentifkasi kontaminan kunci/utama

    *dentifkasi kemungkinan kontaminansi alami yang

    tersisa4ebih baik kemurnian tinggi daripada kurang

    Kontaminan yang mendegradasi ataumenginakti"kan protein atau mengganggu dalam

    analisis sebaiknya dihilangkan sejak aalPeriksa stabilitas protein, minimal kaitannya

    dengan pA dan kekuatan ionik

    eleksi metode pengujian yang cepat dan reliabel.

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    eleksi sumber proteinaterial dapat berasal dari

    $nimal tissuePlant material7iological Buids (e.g. blood, milk, sera)7acteria

    237*'$'+ e9pression&ermentation cultures (yeast, "ungi, bacteria)2ell cultures (animal cells, plant cells, insect cells)

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    P'+*' CKonsentrasi Protein rendah dalam sumber

    alami (natural sources), maka

    0ibutuhkan induksi ekspresi dalam ekspresinya$tau dalam teknik rekombinan diekspresikandalam berbagai system ekspresi.

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    2lassifcation o" proteins bystructural characterisation

    Structural characteristics Examples Comments

    Monomeric Lysozyme, growth hormone Usually extracellular; often havedisulphide bonds

    Oligomeric with identical subunits Glyceralsehyde!phosphatedehydrogenas, catalase, hexo"inase

    Mostly intracellular enzymes, rarely havedisulphide bonds

    Oligomeric with mixed subunits #etussis toxin $llosteric enzymes, different subunitshave different functions

    Membrane bound % peripheral Mitrochondrial $ase, al"aline phosphatise

    'eadily solubilised in detergents

    Membrane bound % intergral #orins, insulin receptors 'e(uires lipid for stability

    Membrane bound % con)ugated Glycoproteins, lipoproteins,nucleoproteins

    Many extracellular proteins containcarbohydrate

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    2lassifcation o" proteins by"unction

    Function Examples

    $mino acid storage *eed proteins +eg gluten-, mil" proteins +eg caesin-

    *tructural % inert .ollagen, "eratin

    *tructural % with activity $ctin, myosin, tubulin

    /inding % soluble $lbumin, hemoglobin, hormones

    /inding % insoluble *urface receptors +eg insulin receptor-, antigens +eg viral coat proteins-

    /inding % with activity 0nzymes, membrane transporters +eg ion pumps

       ,  e   l  a   t   i  v  e

      a   b

      u  n   d  a  n  c  e

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    Protein properties and their eDect ondevelopment o" purifcation strategies

    Sample and target protein properties Influence on purification strategy

    &emperature stability 1eed to wor" rapidly at lower temperatures

     p2 stability *election of buffers for extraction and purification +conditions for ion exchange, affinity orreverse phase chromatography-

    Organic solvents *election of condition for reverse phase chromatography

    3etergent re(uirements .onsider effects on chromatographic steps and the need for detergent removal considerchoice of detergent

    *alt +ionic strength- *election of conditions for precipitation techni(ues and hydrophobic interactionchromatography

    .ofactors for stability or activity *election of additives, p2, salts and buffers

    #rotease sensitivity 1eed for fast removal of proteases or addition of inhibitors

    *ensitivity to metal ions 1eed to add 03&$ or 0G&$ in buffers

    'edox sensitivity 1eed to add reducing agents

    Molecular weight *election of gel filtration media

    .harge properties *election of ion exchange conditions

    /iospecific affinity *election of ligand for affinity medium

    #ost translational modifications *election of group specific affinity medium

    2ydrophobicity *election of medium for hydrophobic interaction chromatography

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     Eields "or multi1step proteinpurifcations

    FGG

    4 Minimal"an tahap purifi"asi

    4 Optimiasi setiap tahap

    4 2atihati terhadap hasil )i"adiperlu"an beberapa tahap

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     +ahap kunci dalampurifkasiemisahkan protein target dari material aal

    emisahkan padatan dari protein yang ada

    dalam supernatanKoncentration protein

    embuang/membersihkan kontaminan untukmeningkatkan kemurnian

    tabilisasi dari target protein

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     +hree phase purifcationstrategy

    #roses final purifi"asi yang ideal mela"u"an preparasi sampel , meliputie"stra"si dan "larifi"asi yang dibutuh"an, melalui ! tahap utama purifi"asi5umlah tahap purifi"asi ditenru"an oleh startegi purifi"asi, "emurnianyang dibutuh"an dan untu" tu)uan apa protein diguna"an

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    $nalisis ProteinPekerjaan yang dilakukan berkaitan dengan

    keperluan dan penentuan hasil selamapurifkasi ditentukan oleh%ntuk tujuan apa protein tersebut

    7ahan aal sumber diperolehnya proteintersebut. Physical studies e.g. 91ray, ',

    nd product - pharmaceuticals

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    $nalysis kemurnianprotein +otal protein

    pecifc Huantifcation

    $ctivity assays7inding assays

    0etection o" impuritiesAP42

    el electrophoresis

    Protein mass spectrometry

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    $ssay method Useful range .omments

     1anoOrange assay 677ng8ml to67ug8ml   •*amples can be read up to six hours later without any loss in the

    sensitivity•Low protein to protein signal variability•3etection not influenced by reducing agents or nucleic acid

    /.$ method +.ureduction-

    79ug8ml to69mgml   •*amples must be read within 67 mins

    • 1ot compatible with reducing agents

    /radford assay +dye binding-

    6ug8ml to 69mg8ml   •#rotein precipitates over time

    •2igh protein to protein signal variability• 1ot compatible with detergents

    Lowry assay 6ug8ml to69mgml   •Lengthy, multistep procedure

    • 1ot compatible with detergents, carbohydrates or reducing agents

    $bsorbance at :7nm 97ng8ml to:mg8ml   •2igh protein to protein signal variability

    •3etection influenced by nucleic acids and other U< absorbingcontaminants

    ethods "or Huantifcation o"proteins in solution

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    Protein Purifcation

    2rude e9tract: breaking cells, by osmosis lysisor homogeniIation.&ractionation: separate proteins into diDerent

    "raction based on siIe o" charge.

    alting out: +he solubility o" proteins isloered at high salt concentration.$mmonium sul"ate (('AJ)K3J).

    0ialysis is a procedure to separate proteins"rom solvents

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     +$A$P P%*&*K$* ($da +ugas)[1] Protein Source: 1. Tissue 2. Microbial cells.

    Jika kita isolasi protein dari jaringan sel, kita harus

    mengisolasi bagian dari sel, misalnya organela• Dapat dilakukan sentrifugasi dengan kecepatan

    bertahapbertingkat

    1. !emecahan sel "#rind tissue "blenderhomogeni$er%

    2. &entrifuge ' the speed determines (hich organellesappear in the pellet and this is based on density"(hich ones (ill pellet first)%

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    4yse 2ellsPhysical&rench pressure cell

    onicationlass beads

    2hemical0etergents

    nIymes

    Aypotonic buDerhttp://.diversifed1eHuipment.com/pics/FFLG.jpg

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    tabiliIe ample

    2ontrol pA%se appropriate buDer

    2ontrol temperatureKeep samples on ice or ork in cold roomPrechill instruments

    Prevent "rothing/"oamingAandle gently.

    aintain concentrated sample

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    tabiliIe ampleProtease inhibitors

    Phenylmethylsul"onyl Buoride (P&)

    4eupeptin

    $protinin

    2hymostatin

    Pepstatin $

    S

    O

    O

    F

    !M6

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    Protein olubilityalting in *ons shield charges and

    allo proteins to "old.

    alting out *ons compete ith ater

    to interact ith sidegroups. Mhen NsaltO ishigh enough, salt inscausing protein to

    precipitate.

    enerally use ammoniumsul"ate to precipitateproteins in the lab.

    :salt;

          s      o        l      u        b        i        l        i       t      y

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    eparate 2ell 0ebris

    2entri"ugationupernate

    Pellet

    &ilter

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    "ig 17%

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    [3] Stabilization

    !roteins are most stable under p0 and ionic

    strength close to physiological conditions.p0+ >., "lysosomal en$ymes, p0 ?%

    I  "ionic strength% @ .1? M

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      [4] Solubilities of Proteins

    Salting In

      4ddition of salt at lo( ionic strength can increase

    solubility of a protein by neutrali$ing charges on the surface

    of the protein, reducing the ordered (ater around the

    protein and increasing entropy of the system.

    Salting out  "&an be used for ractionation)

      Af the concentration of neutral salts is at a high le-el

    "B.1M%, in many instances the protein precipitates.

    This phenomenon apparently results because thee5cess ions "not bound to the protein% compete (ith

    proteins for the sol-ent. The decrease in sol-ation and

    neturali$ation of the repulsi-e forces allo(s the proteins to

    aggregate and precipitate. This effect is called Csalting

    outC.

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    Salting outAmmonium sulfate (half saturated)

    thanol (!"#)•

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      [$] %ial&sis

    • ollo(ing a saltingout step, the solution (ill

    contain a high concentration of salt that can be

    disrupti-e to subseuent chromatographic steps.• The salt can be remo-ed by dialysis ' dialysis

    tubing has pores (ith a specific molecular

    (eight cutoff that allo(s smaller molecules

    "salt% to pass.

    E5change buffer 

      B / times

    Fuffer' large -olume

    Dialysis tubing (ith protein and high salt

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    0ialysis$ "orm o" siIe e9clusionchromatography.%sed to desalt and

    concentrate protein

    samples.

    0ialysis tubing has setmolecular eight cut oD.3nly molecules or ions

    smaller than M23 illmove out o" the dialysis bag.

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    !rotein molecules "red% are retained (ithin the dialysis bag, (hereas

    small molecules "blue% diffuse into the surrounding medium.

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    ['] Searation *hromatograh&

    Makes use of a mobile phase "fluid usually buffer%

    G a stationary phase "usually small beads%.

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    p0 and :salt; dependent• 6eparates by ionic

    charge+ cations G anions

    &ation e5change

      4nion e5change

    Aon e5change

    chromatography

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    Hhere on the p0 scale should the buffer 

    be compared to the pA of the protein)

     This is an e5ample of cation e5change.

    0o( (ill anion e5change (ork)

    &ation e5changer+&Mcellulose &02&**

     

     4nion e5changer+

      &20

    ?

      DE4Ecellulose &02&0

    2I0&

    20

    ?

     

     

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    &hoosing an ion e5changer (ill depend on+

    The pA -alue of the target protein.

    The p0 of buffer used.

     4n "acidic% protein of a pA of ?.

     4t p0 > "phosphate buffer or Tris buffer%,the protein (ill carry a net negati-e charge.⇒ The protein (ill bind to an anion e5changer "DE4E%⇒ Hill be repelled from a cation e5changer "&M%.

    • 4pply the protein mi5ture containing the target protein.• Kemo-e neutral and basic proteins in flo(through.• Keco-er the target protein (ith increasing :salt;.

     

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    Size e+clusion chromatograh&

    ,el filtration chromatograh&

    • &ontains porous beads

    • 6eparates according to si$e and shape• Larger proteins e5cluded

    from the small pores

    • uaternary structure determination,  G Mr  estimation using

    a standard cur-e

      "log Mr  vs elution -olume%

     

    Ⓠ ibrous proteins6pherical vs rodshaped proteins 

    l l i ht ti

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    molecular (eight, nati-emolecular sie-e chromatograh& (gel filtration. molecular

    sizing)

    light scattering

    h&drod&namic (e/uilibrium centrifugation)

    molecular sie-e chromatograh& (gel filtration. molecularsizing)

    light scattering

    h&drod&namic (e/uilibrium centrifugation)

    mi+

    largemoleculesfirst

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    ,el 0iltration *hromatograh& 4 mi5ture of proteins in a small -olume is

    applied to a column filled (ith porous beads. Fecause large proteins

    cannot enter the internal -olume of the beads, they emerge sooner than do

    small ones.

    4ffi it h t h

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    • 6eparates by specific interactions

    • &ontains a ligand+

    en$yme substratereceptor hormoneantigen antibody"0is%

    9 protein ' Ii2

     4ffinity chromatography

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    $Qnity

    2hromatographyobile phase

    %sually liHuid

    tationary phase

    eceptor bound toinert bead

    Ammunoaffinity column

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    http://F.Hiagen.com/products/protein/images/fgR42RBe9ibleRimmobiliIation.gi" 

    glutathione

    ,S tag

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     +hin 4ayer

    2hromatography

    elative "ront value

    "  S distance traveled by substancedistance traveled by solvent

    Aigh " value S nonpolar substance4o "  value S polar substance

    *rigin

    6ol-ent

    front

    Distance tra-eled

    by substance

    Distance tra-eled

    by sol-ent

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    everse Phase

    2hromatography

    obile phase

    Polar liHuid

    tationary phase'onpolar liHuid immobiliIed

    on inert solid

    Aigh " value S polar substance

    4o "  value S nonpolar substance

    *rigin

    6ol-ent

    front

    Distance tra-eled

    by substance

    Distance tra-eled

    by sol-ent

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    Aigh Per"ormance 4iHuid

    2hromatographyobile phase

    4iHuid

    tationary phasemall diameter particles

    packed into column.

    Pressure is reHuired topush liHuid throughcolumn.

    $dvantages7etter resolving poer&aster

    http://.aters.com/atersdivision/2ontent0.asp5atersitS601>4+7A

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    Aigh Per"ormance 4iHuid

    2hromatography

    http://academic.sun.ac.Ia/sa"/units/aaa/images/imageJRl.jpg

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    Electrophoretic 4nalysis6eparates according to si$e and charge concentration

    Matri5 is polyacrylamide "proteins% or agarose "nucleic acids%

     6D6!4#E "polyacrylamide gel electrophoresis%+ 

    uses sodiumdodecyl sulphate "6D6% to coat the proteins

    to gi-e them all eui-alent "negati-e% charge concentration

     ' proteins can then separate by M.H. only

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    Pol&acr&lamide ,el lectrohoresis

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    Pol&acr&lamide ,el lectrohoresis The negati-ely charged 6D6 "sodium

    dodecyl sulfate%protein comple5es migrate in the direction of the anode, at

    the bottom of the gel. The sie-ing action of a porous polyacrylamide gel

    separates proteins according to si$e, (ith the smallest mo-ing most rapidly.

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    ti ti th l l M i ht "

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    stimating the olecular Meight o" aProtein

    Asoelectic focusing

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    •  4mpholytes are lo( M.H. organic acids and bases

    that distribute along the electric field across the gel.

    •  Each protein of a mi5ture distributes across the gelaccording to their pA.

    soelctric focusing can be described as electrophoresis in a p0 gradient

    set up bet(een a cathode and anode (ith the cathode at a higher p0 than

    the anode. Fecause of the amino acids in proteins, they ha-e amphotericpropertites and (ill be positi-ely charged at p0 -alues belo( their pA and

    negati-ely charged abo-e. This means that proteins (ill migrate to(ard

    their pA. Most proteins ha-e a pA in the range of ? to 7.?.

    Nnder the influence of the electrical force the p0 gradient (ill be

    established by the carrier ampholytes, and the protein species migrate and

    focus "concentrate% at their isoelectric points. The focusing effect of theelectrical force is counteracted by diffusion (hich is directly proportional to

    the protein concentration gradient in the $one. E-entually, a steady state is

    established (here the electrokinetic transport of protein into the $one is

    e5actly balanced by the diffusion out of the $one.

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    2D electrophoresis

    !erhaps certain proteins ha-e

    identical pAs or molecular (eights

    1. 6eparate by AE ' 1st dimension

    2. 6eparate by 6D6!4#E ' 2nd dimension 

    this gi-es a second dimension to the analysis

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    *soelectric &ocusingp* o" a protein: net

    chargeSG

    $ pA gradient isestablished byalloing a mi9ture o"organic acids andbases (ampholytes).Protein migratesuntil it reaches thepA that matches itsp*

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    onitoring Progress o"

    Purifcation Protocol!otal protein 'mg(

    Tuantity o" protein present in "raction

    !otal a)ti*ity 'units of a)ti*ity(

    %se a portion o" sample to determine activity.

    ultiply activity by total volume to determinetotal activity.

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    onitoring Progress o"

    Purifcation Protocol+pe)i) a)ti*ity 'units of a)ti*ity,mg(

     +otal activity

     +otal protein

    yield  measure o" activity retaineda"ter each step in procedure.

    6.4. @

    Q yield @Total acti-ity at particular step

    Total acti-ity of initial e5tract

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    onitoring Progress o"

    Purifcation Protocol

    Puri)ation le*el  easure o" increase in

    purity o" protein throughout procedure.

    !urification @

    6pecific acti-ity at particular step

    6pecific acti-ity of initial e5tract

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    01P$odium dodecyl

    sul"ate polyacrylamidegel electrophoresis

    eparation based onmolecular mass.

    2oat samples ith

    0 to give uni"ormcharge to mass ratio.akes all proteins

    negatively charged.

    http://.activemoti".com/images/products/nitedRpchromoRgel.jpg

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    $40*1+3&4aser displaces

    sample into

    ioniIation chamber.

    *ons travel throughelectrical feld.

    Aeavier ions travelmore sloly.

    http://oregonstate.edu/instruction/bbJ>G/stryer/chGJ/lide.jpg

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    *soelectric &ocusing

    eparationbased oncharge.

    2an be used toe9perimentallydetermine p*.

    http://."ood.rdg.ac.uk/online/"sJG/lectureJ/lJa.gi" 

    anode cathode

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    Sray &rystallography Electrons in crystal scatter 5rays to produce an image.

    ourier transformation is used to con-ert ra( data into /D structure.

    Protein /+3 setelah pemanasan 15 menit

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    200 KDa

      150 KDa

    120 KDa

      100 KDa  5 KDa

    !0 KDa

      "0 KDa

     50 KDa

      #0 KDa

      $0 KDa

    25

    KDa  20 KDa

      7 % %8" %9" %!" %1""

    'suhu kultur 0(

    D @ crude protein "cd%

    D> @ cd dipanaskan > derajat &elsius

    D7 @ cd dipanaskan7 derajat &elsius

    D8 @ cd dipanaskan 8 derajat &elsius

    D1 @ cd dipanaskan 1 derajat

    &elsius

    #rodu" #urifi"asi #rotein dari >solat 3*! dalam rang"ad t" 31$ li t t bil

  • 8/18/2019 Purifikasi Protein

    77/80

    #ambar Profl hasil pemurnian 0'$ polimerase dari isolat 0Lpada 01P$ F= dengan pearnaan 2oomassie Brilliant BlueR250. S penanda protein, F S protein kasar, S protein kasardipanaskan pada suhu !>oc selama F> menit, L S Protein produkkromatograf penukar ion, J S Protein kromatograf gel fltrasi,

    S protein yang hilang

    mendapat"an 31$ polimerase termostabil

    %rodu& amplifi&asi D'( dalam %C) oleh protein dari

    i l t DS$

  • 8/18/2019 Purifikasi Protein

    78/80

    #ambar Produk amplifkasi 0'$ gen caree dalam plasmid P+* menggunakan0'$ polimerase produk pemurnian. P2 menggunakan primer universal FL Forward

     Amplication Primer , dan primer FL Reverse Amplication Primer . lektro"oresisdilakukan pada gel agarose F= (g/v). S Penanda 0'$ 7FL/Hinf *, F S ega i91oyal, S Protein kasar, L S Protein kasar dipanaskan !>o2 F> menit, J S 2ampuranprotein dalam "raksi #1F# produk kromatograf penukar ion, > S protein dalam "raksi>J produk kromatograf gel fltrasi, S +anpa protein pengganti Taq polimerase, ! S

    tanpa template 0'$.

    isolat DS$

  • 8/18/2019 Purifikasi Protein

    79/80

    Konsentrasi *S(/D

  • 8/18/2019 Purifikasi Protein

    80/80

    +,g-ml./D55nm

    7 7: 779? 76?9

    76@?6@ 7!!!: 7@A@@? 6!?!

    &ahap purifi"asi Berapatan Opti" padaC9D9 Badar protein +Eg8ml-

    #rotein "asar #rotein yangdipanas"an suhu A9o.69 menit

    Fra"si 67 hasil"romatografi penu"arionFra"si 9? hasil"romatografi gel filtrasi

    7,!7

    7,!69

    7,:9@

    7,79D

    ,??

    @,9

    9,?6

    7,@6