Gramicidin perforated patches

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biophysics.tushar
biophysics.tushar's picture
Gramicidin perforated patches

 I have been using Gramicidin perforated patches to study neuronal chloride dynamics.
However, After forming Giga seal I need to wait ~45 min some times even more than that to get membrane resistance ~40 M ohom. 
I tried different concentrations of Gramicidin and DMSO (solvent to dissolve gramicidin) too.
Can anyone give me any suggestion. How can I reduce penetration time?

zp
zp's picture
i believe a long wait with

i believe a long wait with gramacidin is normal. i too have to wait about 40min when using gramicidin. if you continue to increase the concentration you will increase your chances of spontaneously rupturing the membrane (Kyrozis & Reichling, 1995). also note that the patched membrane continues to "creep up" the pipette for several minutes (10-20min depending on the size and shape of the pipette) after seal formation (this can be observed by imaging the tip of your electrode and patching a fluorescent cell). as a result, with time, more of the membrane is exposed to the perforating agent. as such, this "creeping" imposes a minimum 20-30min wait period before the perforation stablizes, regardless the nature or concentration of the perforating agent being used.