Role of EDTA in protoplast lysis?

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    • #215
      silvie
      Participant

      Hello together! I am isolating vacuoles and for that purpose I first purify protoplast of Arabidopsis leave mesophyll. Afterwards I have to lysate the protoplasts. The lysis buffer contains EDTA. Can anyone tell me what the exact function of EDTA is in protoplast lysis? For certain causes (measurement of metals afterwards) I want to use a buffer without EDTA, but the lysis was worse without EDTA.
      ??? Can somebody help?

    • #18688
      biostudent84
      Participant

      EDTA solution was fed to the petioles, which is known to prevent callose sealing of sieve tubes.

      http://wwwuser.gwdg.de

      That is the only reference I have been able to find. What class and level work are you doing?

      Kyle

    • #18694
      silvie
      Participant

      @biostudent84 : Thank you for your reply.
      This project is part of my PhD work, which I have just started a few weeks ago in Stuttgart (Germany). I asked that question to nearly all people I know, but nobody seems to know why protoplast lysis works better with EDTA than without. Seems that some metals, if not complexed by EDTA, have some effect on the lysis procedure. But I cannot imagine which exact mechanism is responsible for that, to avoid this problem…
      So, still the same question: if anybody can tell me, I would be glad!!
      I also checked lots and lots of publications. Either I am to stupid to find this 😳 or nobody published something about it, or it is so simple that everybody thinks it is not worth mentioning… ❓

    • #18695
      biostudent84
      Participant

      PhD work? I wish you luck with that, my friend. I am a mere undergraduate, so I am afraid I would be unable to help you. There are certain parts to our library that are restricted to graduate students only. Wish I knew why =(

      It might simply be one of those things that we have to accept and not understand why. Although if you are allowed access to the materials to do the experiment, maybe you could do your doctoral dissertation on it?

      Kyle

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