A Little Help Please

Dear Readers… I suspect that some of you may be electrophysiologists and that you may be able to help us out.  We dabble in whole cell patch clamp and are transitioning to doing gramicidin perforated patch for a project.  We’re having a very hard time getting it to work (whole cell is no problem for us).  We suspect that our gramicidin is the problem (we got it from Sigma G5002).  Does anyone know a quality source of gramicidin for perforated patch.  Company and part number would be helpful.  Also, if you have a detailed protocol we’d appreciate getting it by email (see the about page for my email).  Thanks in advance to anyone that can offer some advice.

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3 responses to “A Little Help Please

  1. Pingback: Blogrolling TRP channels « JUNIORPROF

  2. Well, I have checked with my two sources who have done some gramicidin perforated patch, and both have used the Sigma gramicidin, presumably the G5002 (a mixture of A,B,C and D forms.

    Typically it’s diluted in DMSO, and can be used at pretty high concentrations (~100 microg/mL). Sonication doesn’t seem to be required to solublize the gramicidin, though people did typically remake the internal with fresh stock gramicidin (every couple hours or so).

    Also, people have not front filled the pipettes, so the formation of electrical access is pretty rapid (<5-10 min).

    Sorry there is such a lack of details; people have either done these expts some time ago, or never published their results so have less firm protocols written about it. I suggested that these folks should start their own personal wikis for these kinds of info (I have one myself) but I don’t think they have.

    One thing I might try for a different supplier of the gramicidin, would be Calbiochem. They sell a pure gramicidin A, which might be easier to troubleshoot.

  3. Thanks Nat, this is useful stuff for us.

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