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Affinity chromatography
1.
AAffffiinniittyy CChhrroommaattooggrraapphhyy AAuuddrreeyy
CChheeuunngg MMiicchhaaeell KKwwoonngg MMaarrcc--AAnnddrree LLyynncchh Image source: Bio-Rad Laboratories
2.
FFuunnccttiioonn iinn pprroocceessss
RReemmoovvaall ooff ccoonnttaammiinnaanntt pprrootteeiinnss UUttiilliizzeess aaffffiinniittyy ooff pprrootteeiinn--AA ffoorr tthhee MMaabb ttoo ppuurriiffyy tthhee pprroodduucctt.. HHiigghh eeffffiicciieennccyy –– 9900%% PPrrootteeiinn--AA eeffffeeccttiivvee ffoorr mmoonnoocclloonnaall aannttiibbooddyy ppuurriiffiiccaattiioonn..
3.
HHooww iitt wwoorrkkss
11.. EEqquuiilliibbrraattee –– aadddd mmiixxttuurree ttoo bbaattcchh ttoo bbiinndd ttaarrggeett mmoolleeccuullee ((MMaabb)) 22.. WWaasshh oouutt mmiixxttuurree –– rriidd ooff iimmppuurriittiieess 33.. AAdddd wwaasshh bbuuffffeerr 44.. RReemmoovvee wwaasshh bbuuffffeerr 55.. AAdddd eelluuttiioonn bbuuffffeerr 66.. RReemmoovvee eelluuttiioonn bbuuffffeerr ccoonnttaaiinniinngg ttaarrggeett mmoolleeccuullee 77.. RReeggeenneerraattiioonn ooff oorriiggiinnaall ccoonnddiittiioonnss Image source:Wikipedia
4.
AAlltteerrnnaattiivveess • PPrrootteeiinn
GG –– ccaann hhaavvee hhiigghheerr aaffffiinniittyy ffoorr cceerrttaaiinn mmoolleeccuulleess hhoowweevveerr iitt iiss ggeenneerraallllyy mmoorree eexxppeennssiivvee.. • PPrrootteeiinn LL -- uusseedd ffoorr ppuurriiffyyiinngg mmoonnoocclloonnaall aannttiibbooddiieess ffrroomm iimmppuurriittiieess.. CCaann bbee oorrddeerreedd aass rreeaaddyy--ttoo--uussee pprreeppaacckkeedd ddeevviicceess ffoorr iissoollaattiioonn aanndd ppuurriiffiiccaattiioonn ooff iimmmmuunnoogglloobbuulliinn.. • MMeelloonn GGeell SSuuppppoorrtt -- bbiinnddss nnoonn--aannttiibbooddyy sseerruumm pprrootteeiinnss ((eexx.. aallbbuummiinn aanndd ttrraannssffeerrrriinn)) wwhhiillee aalllloowwiinngg tthhee aannttiibbooddyy ttoo ffllooww tthhrroouugghh iinn aa mmiilldd bbuuffffeerr.. OOffffeerrss aa ggeennttllee wwaayy ttoo ppuurriiffyy aannttiibbooddiieess ffrroomm sseerruumm.. Source: www.pierce.net
5.
CCuurrrreenntt ddeessiiggnn PPaarraammeetteerrss
ooff iinntteerreesstt ffoorr ooppttiimmiizzaattiioonn:: VVoolluummee llooaadd,, mmaassss llooaadd,, ffllooww rraattee,, rreessoolluuttiioonn,, rreeccoovveerryy,, bbeedd ccoonnffiigguurraattiioonn,, ppuurriittyy aanndd ccoosstt..
6.
PPaarraammeetteerr iinnvveessttiiggaattiioonn
DDiiffffiiccuulltt ttoo vvaarryy tthhee yyiieelldd lliikkeellyy dduuee ttoo aann aallrreeaaddyy hhiigghh aaffffiinniittyy ffoorr MMaabb.. IInnvveerrssee rreellaattiioonnsshhiipp bbeettwweeeenn bbaattcchh ttiimmee aanndd lliinneeaarr vveelloocciittyy.. EEffffeecctt ooff lliinneeaarr vveelloocciittyy oonn MMaabb//aacceettiicc aacciidd rraattiioo ffoouunndd ttoo bbee nneegglliiggiibbllee.. IInnccrreeaassee iinn ccoolluummnn hheeiigghhtt rreessuulltteedd iinn sslliigghhtt iinnccrreeaassee iinn bbaattcchh ttiimmee.. IInnccrreeaassee iinn hheeiigghhtt ddeeccrreeaasseedd MMaabb//aacceettiicc aacciidd rraattiioo –– ccoonnssiisstteenntt wwiitthh tthheeoorryy tthhaatt ccoolluummnnss tteenndd ttoo bbee sshhoorrtteerr aanndd wwiiddeerr iinn ssccaallee--uupp pprroocceessss..
7.
PPaarraammeetteerr iinnvveessttiiggaattiioonn
DDiiffffiiccuulltt ttoo vvaarryy tthhee yyiieelldd lliikkeellyy dduuee ttoo aann aallrreeaaddyy hhiigghh aaffffiinniittyy ffoorr MMaabb.. IInnvveerrssee rreellaattiioonnsshhiipp bbeettwweeeenn bbaattcchh ttiimmee aanndd lliinneeaarr vveelloocciittyy.. EEffffeecctt ooff lliinneeaarr vveelloocciittyy oonn MMaabb//aacceettiicc aacciidd rraattiioo ffoouunndd ttoo bbee nneegglliiggiibbllee.. IInnccrreeaassee iinn ccoolluummnn hheeiigghhtt rreessuulltteedd iinn sslliigghhtt iinnccrreeaassee iinn bbaattcchh ttiimmee.. IInnccrreeaassee iinn hheeiigghhtt ddeeccrreeaasseedd MMaabb//aacceettiicc aacciidd rraattiioo –– ccoonnssiisstteenntt wwiitthh tthheeoorryy tthhaatt ccoolluummnnss tteenndd ttoo bbee sshhoorrtteerr aanndd wwiiddeerr iinn ssccaallee--uupp pprroocceessss..
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