Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Polyclonal antibody production using SDS-PAGE gel fragment


  • Please log in to reply
3 replies to this topic

#1 KOBUS

KOBUS

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 01 December 2011 - 04:14 AM

Hi Guys/Gals

I need to produce antibodies for CO-IP, but my protein of interest is produced as inclusion bodies and many expression optimisation / refolding attempts have failed. Do any of you know of a protocol that uses SDS-PAGE gel fragment directly for antibody production? I've seen that certain antibody production companies only require you to send them a dried SDS-PAGE band, but i cant manage to find instructions! Will the presence of SDS affect the immunization? Surely?

Thanks

#2 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,811 posts
136
Excellent

Posted 01 December 2011 - 09:22 AM

the sds present will have no significant effect on the immunization. keep in mind that you will be generating antibodies to denatured protein and may not recognize native protein.
talent does what it can
genius does what it must
i do what i get paid to do

#3 protolder

protolder

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 293 posts
9
Neutral

Posted 01 December 2011 - 11:42 PM

Hola,to me the main requirement to extract protein of a gel is don´t fix stain because protein remains fix to acrylamide and it isn´t easy extract it. (I don´t know if people immunizes with an blue suspension of acrylamide). Some years before, we run preparative gels with a bit of Comassieblue in the upper buffer and after run we cut band, pass throught a 10-20 ml srynge and homogenize with a tisssue blade homogenizer extracting of the suspension some times untill the supernatant were less blue. after dyalisis and liophilization and reconstitution you will inject a blue solution to rabbits or mice. Buena suerte

#4 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,811 posts
136
Excellent

Posted 02 December 2011 - 12:15 PM

we've injected fixed, stained and mashed up gel slices and had excellent results (we do wash out the methanol/acetic acid prior to homogenizing the gel slice in pbs).
talent does what it can
genius does what it must
i do what i get paid to do




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.