Elektroforetische scheiding van eiwitten

Biology

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Summary

In deze video tonen we een methode voor de elektroforetische scheiding van eiwitten met behulp van poly-acrylimide gelelektroforese (PAGE).

Cite this Article

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Chakavarti, B., Chakavarti, D. Electrophoretic Separation of Proteins. J. Vis. Exp. (16), e758, doi:10.3791/758 (2008).

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Abstract

Elektroforese wordt gebruikt om complexe mengsels van eiwitten (bijvoorbeeld van cellen, subcellulaire fracties, kolom breuken of immunoprecipitaten) te scheiden, om subunit composities te onderzoeken en om de homogeniteit van eiwit monsters te controleren. Het kan ook dienen om eiwitten te zuiveren voor gebruik in verdere toepassingen. In polyacrylamidegelelektroforese, eiwitten migreren als reactie op een elektrisch veld door de poriën in een polyacrylamide gel matrix; poriegrootte afneemt met toenemende acrylamide concentratie. De combinatie van poriegrootte en eiwit lading, de grootte en de vorm bepaalt de migratie snelheid van het eiwit. In deze unit, is de standaard Laemmli methode beschreven voor discontinue gelelektroforese onder denaturerende omstandigheden, dat wil zeggen, in het bijzijn van natriumdodecylsulfaat (SDS).

Protocol

De volledige tekst protocol voor deze experimentele aanpak is beschikbaar in Current Protocols in Molecular Biology .

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Comments

10 Comments

  1. Hi nice demo but I did not understand why the presenters kept on showing an agarose gel electrophoresed DNA gel in between as a representation instead of a protein gel.

    Reply
    Posted by: Anonymous
    June 15, 2008 - 1:11 PM
  2. SYPROorange and SYPROruby staining - two fluorescent-based protein detection methods - can appear like ethidium bromide stained DNA gels.  So those gels you are speaking of may actually be protein gels. 

    Reply
    Posted by: Anonymous
    June 16, 2008 - 9:34 AM
  3. Yeah possible,  but then why do we see only a single band.I guess SYPRO detection methods would detect the entire protein panel and not specific bands. unless otherwise they are detecting some specific enzyme activity using flourescent detection methods.

    Reply
    Posted by: Anonymous
    June 16, 2008 - 9:52 AM
  4. We loaded BSA.

    Thanks.

    Reply
    Posted by: Anonymous
    June 17, 2008 - 2:12 AM
  5. Please not that we showed polyacrylamide gel electrophoresis and not agarose gel electrophoresis.

    Thanks.

    Bulbul Chakravarti

    Reply
    Posted by: Anonymous
    June 17, 2008 - 2:08 AM
  6. why cannot i see the video...? pls help me..

    Reply
    Posted by: Anonymous
    October 10, 2008 - 3:35 AM
  7. i can see it now..thanks...

    Reply
    Posted by: Anonymous
    October 10, 2008 - 4:50 AM
  8. my network is poor, I can not enjoy the video

    Reply
    Posted by: Huang C.
    March 15, 2009 - 6:30 AM
  9. Hello, Please shoot me an email at nikitab@jove.com and we'll figure something out for you. Cheers, Nikita

    Reply
    Posted by: Anonymous
    March 22, 2009 - 10:26 PM
  10. Hi, thanks what can I download your video ? please help me

    Reply
    Posted by: violet s.
    January 14, 2012 - 10:35 AM

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