<p class="jove_content">Shrinking solution recipe is from the lab of R.W. Aldrich. We thank the following funding agencies and foundations for support: National Institutes of Health, National Science Foundation, American Heart Association, Muscular Dystrophy Association, the Donald B. and Delia E. Baxter Foundation, the Klingenstein Fund and the McKnight Endowment for Neuroscience.</p>

<p class="jove_step">Part 1: Removing the vitelline membrane</p>
<ol>
<li>Prepare two sets of forceps. We prefer No. 5 forceps, where one set has been slightly sharpened with a file. Also prepare a glass transfer pipette by trimming and fire-polishing a 7" Pasteur pipette.</li>
<li><em>Recommended:</em> To prevent the oocytes from slipping, cut a circle of grid (1 mm squares) and glue into the bottom of a 60mm Petri dish. This dish can be reused indefinitely if kept clean between uses.</li>
<li>Fill the Petri dish halfway with hyperosmotic solu...

<ol>
	<li>Neher, E., Sakmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-channel+currents+recorded+from+membrane+of+denervated+frog+muscle+fibres.">Single-channel currents recorded from membrane of denervated frog muscle fibres.</a> <em style='font-style: italic'>Nature</em>. <b>260</b>, 799-802 (1976).</li><li>Sackmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>Single-channel Recording</em>. , (1983).</li></ol>

<p class="jove_content">There are many parameters of electrophysiological recording not discussed here. Rig setup, system noise management, channel expression, and recording protocols are all also critical to good experimental results.</p>
<p class="jove_content">In our experience, these are the most critical parameters for forming reliable seals: high quality oocytes, removing the vitelline membrane quickly (a.k.a., "peeling"), use newly pulled pipettes protected from dust, smooth pipettes, positive pressure applied when entering the bath sol...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Patch Clamp Amplifier & Software</td>
<td>Instrument</td>
<td>HEKA or Axon</td>
<td>EPC-10</td>
<td>Or other similar device (e.g. HEKA EPC-9 or Axopatch 200B)</td>
</tr>
<tr>
<td>No. 5 forceps (two pairs)</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Oocyte shrinking solution</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Ingredient                            In mM
N-methyl-D-glucamine     220
HEPES                                   10
MgCl2                                       1
EGTA                                       10
Aspartic Acid                        220
KCl                                             2
pH to 7.4 w/ N-methyl-D-glucamine</td>
</tr>
<tr>
<td>woven mesh</td>
<td>800 um</td>
<td>Spectrum Laboratories INC</td>
<td>146481</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
	

patch clamp recording of ion channels expressed in  xenopus oocytes

<p class="jove_content">Since its development by Sakmann and Neher <sup>1, 2</sup>, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells.  This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis.  Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes.  This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording).  We focus on techniques to maximize the quality of oocyte preparation and seal generation.  With all factors optimized, this technique gives a probability of successful seal generation over 90 percent.  The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.</p>

Watch this Scientific Journal Video about Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes at JoVE.com

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

<p class="jove_content">This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes.  It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.</p>

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Research

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The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify ...

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The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...