3protein purification and characterization techniques

7/17/2019 3protein Purification and Characterization Techniques

1/19

Protein PurificationProtein Purificationand Characterizationand Characterization

TechniquesTechniques

Nafth Abu Tarboush, DDS, MSc, PhD

[email protected]

www.acebook.com/natarboush
mailto:[email protected]://www.facebook.com/natarboushhttp://www.facebook.com/natarboushmailto:[email protected]


2/19

Extracting Pure Proteins from CellsExtracting Pure Proteins from Cells Purifcation techniues ocus main!" on si#e $

char%e The frst ste& is homogenization '%rindin%,

Potter()!*ejhem homo%eni#er, sonication,ree#in% and thawin%, deter%ents+

Diferential centriugation '-- g:unbroken ce!!s $ nuc!ei 0,--- g:mitochondria --,--- g: ribosomes and

membrane ra%ments+


3/19

Salting in & outSalting in & out Are &roteins so!ub!e1 2 "es, to which !imit1

Sa!t stabi!i#es the *arious char%ed %rou&s on a&rotein mo!ecu!e and enhance the &o!arit" owater, thus attractin% &rotein into the so!utionand enhancin% the so!ubi!it" o &rotein

Ammonium su!ate is the most common rea%entto use at this ste&

This techniue is im&ortant but resu!ts arecrude


4/19

DialysisDialysis

Princi&!e o di3usion 4once&t o M5 cut6o3

Pure *s. crude


5/19

Column ChromatographyColumn Chromatography 7reek chroma, 8co!or,9 and graphein, 8to write9

2s it just or co!ouru! &roteins1 4hromato%ra&h" is based on two &hases: stationar" $

mobi!e

5hat are the di3erent kinds1


6/19

Size-exclusion chromatographySize-exclusion chromatography

Gel-filtration chromatographyGel-filtration chromatography

Se&aration on the basis o si#e 'M5+

Stationar" 'cross6!inked %e! &artic!es+:consist o one o two kinds o &o!"mers thestis a carb. &o!"mer 'e;. dextran or

agarose); oten reerred to b" Se&hade;and Se&harose. The


7/19

Molecular-sieveMolecular-sieve

chromatographychromatography


8/19

Affinity chromatographyAffinity chromatography 2t has s&ecifc bindin%

&ro&erties The &o!"mer 'stationar"+ is

co*a!ent!" !inked to a ligandthat binds s&ecifca!!" to thedesired &rotein

The bound &rotein can bee!uted b" addin% hi%h conc. othe so!ub!e !i%and

Protein(!i%and interaction can

a!so be disru&ted with achan%e in &> or ionic stren%th

4on*enient $ &roducts are*er" &ure 'Anti%en6antibod",>is6ta%, 7ST6Ta%+


9/19

Ion-exchange chromatographyIon-exchange chromatography 2nteraction based on net char%e $ is !ess s&ecifc

?esin is either ne%ati*e!" char%ed 'cation exchanger+or &ositi*e!" char%ed 'anion exchanger+

=u3er eui!ibration, e;chan%e resin is bound tocounter6ions. A cation6e;chan%e resin is usua!!" boundto Na or ions, and an anion e;chan%er is usua!!"bound to 4!(ions

Proteins mi;ture !oadin%

)!ution 'hi%her sa!t concentration+


10/19

ElectrophoresisElectrophoresis

=ased on the motiono char%ed &artic!esin an e!ectric fe!d

Macromo!ecu!esha*e di3erin%mobi!ities based ontheir char%e, sha&e,and si#e

The most commonmedium is a&o!"mer o a%aroseor acr"!amide


11/19

Agarose vs. PAGEAgarose vs. PAGE A%arose 'nuc!eic acids+, PA7)

'&roteins+

2n PA7): SDS or NB6SDSC4>D'4>


12/19

IsoelectricIsoelectric

focusingfocusing

Proteins ha*edi3erent isoe!ectric

&oints 7e! &re&ared with a

&> %radient &ara!!e!to e!ectric6fe!d

%radient Two6dimensiona! %e!

e!ectro&horesis '


13/19

Immunoassays Western blotImmunoassays Western blot From %e! to a membrane 'nitroce!!u!ose or

&o!"*in"!idene diGuoride, PHDF+

Detection:

4o!orimetric: en#"mes bound to


14/19

Immunoassays - ELISAImmunoassays - ELISA )n#"me6Jinked 2mmunosorbent Assa"

Detect $ uanti" substances' &e&tides, &roteins, antibodies $hormones+

Ksua!!" done in L6we!! &o!"st"rene&!ates '&assi*e!" bind antibodies and&roteins+

A&!!ication:

Screenin% '>2H, >e&atitis =$4+

>ormones '>47, J>, TS>, T, T+

'7reen,&ositi*e+

'Noco!or,

ne%ati*e+


15/19

Protein sequencing -Protein sequencing -

Edman MethodEdman Method Ste& : how much and which amino acids

are in*o!*ed

Hydrolysis 'heatin% >4!+ & Separation'ion6e;chan%e chromato%ra&h" or b" highperormance liquid chromatography

H!"#)


16/19

Protein sequencing -Protein sequencing -

Edman MethodEdman Method Ste&


17/19

Tr"&sin: 4!ea*es @ 46termina! o '+ char%edside chains

4h"motr"&sin: 4!ea*es @46termina! o aromatics

4N=r: 4!ea*es @ 46termina! o 2NT)?NAJ

methionines


18/19

Protein sequencing Protein sequencing

Mass SpectrometryMass Spectrometry

ass*charge ratio


19/19

Protein sequencing Protein sequencing

prediction from DNA & RNAprediction from DNA & RNA

2 the seuence o the %ene is known, this is

*er" eas" 2 the seuence o the %ene is unknown

'new!" iso!ated &roteins+1 Seuence a short

se%ment, com&!ementar" ?NA, iso!atem?NA, P4?, %ene seuencin%

3protein purification and characterization techniques

Documents