Dear Kirankumar,

I have not patched vesicles before but from my 20 years of experience with this method I would recommend you to use either perforated patch-clamp or first sealing the membrane and then breaking the membrane (electroporation) with voltage pulses instead of suction. I have been successfully using this method with human iPSC derived various cell types in culture, retina and brain slices (see publications listed below). If your vesicles are small you may need slightly smaller tip glass electrodes of 5-8 megohm (preferably polished) instead of usual ~3 megohm used for cells. Basically once you form gigaseal with the membranes then you need to apply incrementing levels of voltage steps at 5-10mV steps and 0.5 to 1 s duration. You do not have to use Neurobiotin in your pipette solution. For details see my publications below.

best wishes,

Refik

Article Semi-loose seal Neurobiotin electroporation for combined str...

Article Neurobiotin Electroporation for Combined Structural and Func...

Hi, Kirankumar

Here you have three papers which might help you in undertaking patch-clamp studies in GUVs. Best wishes and good luck. Francisco

Distribution, lateral mobility and function of membrane proteins incorporated into giant unilamellar vesicles.Doeven MK1, Folgering JH, Krasnikov V, Geertsma ER, van den Bogaart G, Poolman B.  Biophys J. 2005 Feb;88(2):1134-42. Epub 2004 Dec 1.

 Monitoring and quantifying the passive transport of molecules through patch-clamp suspended real and model cell membranes. Messina P, Lemaître F, Huet F, Ngo KA, Vivier V, Labbé E, Buriez O, Amatore C. Angew Chem Int Ed Engl. 2014 Mar 17;53(12):3192-6

A simple method for the reconstitution of membrane proteins into giant unilamellar vesicles. Varnier A1, Kermarrec F, Blesneac I, Moreau C, Liguori L, Lenormand JL, Picollet-D'hahan N. J Membr Biol. 2010 Feb;233(1-3):85-92.