For years innocent readers thought they had escaped...
A horror so horrible it inspires screams...of horror
It is...the...long awaited post on how to measure a junction potential!!
It's clear from the search terms that bring people here, plenty of folks are curious about this topic. The "What is a Liquid Junction Potential" post is one of the most read. So allow me to finally follow up with how to actually measure it. Although there are the standard descriptions about junction potentials in various texts and bibles of the field, a step by step 'how-to' is lacking. Seeing as I bridge these two in real life lab,why not here?
Ok, you have some internal solutions, and you want to report the correct voltages in your paper.
You've got to account for the junction potential between the internal solution and the bath solution present when you first stick in the pipette. Now, you can calculate it if you want; there's a program built into Clampex. But, are you sure that the constants are correct for what's in your solution, especially if it has something weird in it? TEA-methanesulfonate for example. Or NMDG-aspartate - a killer internal for recording sodium current by the way. Are you even sure you made the solution correctly?
It's easy, so measure it!
When I measure junction potentials, I follow the advice of my old granpappy, as discussed in E. Neher, Methods Enzymol. 1992;207:123-31.
Here's what you want to do:
1) Get your stuff together: battery powered chlorider (don't tell me you're using bleach - GAH!!), silver wire, 3 M KCl, 5+ mL of each of the internal solutions, plus a few mL of the typical bath solution, a few patch pipettes.2) Make a 'flowing KCl' bridge, by taking a patch pipette, breaking off most of the tip, and filling it with 3 M KCl. Chloride a silver wire and use this as the bath ground. The high concentration of KCl, and the similar mobility of K+ and Cl- will help keep the junction potential at this electrode constant, even as the bath solutions changes. I fashioned a little jig to hold the bath pipette (on left below, a piece of plexiglass on a piece of white teflon).
3) Rechloride your silver wire on the headstage, fill a normal resistance patch pipette with one of the solutions. If you're measuring only a single internal/bath pair, then you can put the internal in the pipette. But since setting this up is somewhat annoying, I often measure multiple internals relative to the same bath solution. In that case, I put the bath solution in the pipette.
4) Whatever solution you put in the pipette, add it to the bath.5) Put your amplifier in current clamp mode (slow versus fast doesn't matter), set the 'meter' to 'Vm', make sure that there's no external command signal coming into the amplifier.
6) lower pipette in
to solution.7) Use the "pipette offset" potentiometer until the meter reads zero mV. Wait a minute or so; if the voltage drifts by more than a few tenths of a mV, then you might need to rechloride the wires.
8) Completely exchange the bath solution with the solution to be measured.
9) Read the meter, which shows the junction potential.
10) Replace the bath with the same solution as in the pipette, check that Vm is back close to zero. After that you can measure any other solutions.
11) Now, to get the total transmembrane voltage in your experiments, you have to add your command voltage (or recorded in the case of a current clamp expt) to the measured junction potential. NOTE: If you switched the bath and pipette locations, then you MUST reverse the polarity. So, in the 8.2 mV above, that's actually a junction potential of -8.2, and is added to the command (e.g., a step from -80 to 0 in Clampex becomes -88 to -8 mV). If you're an anal retentive scientist *whistles innocently*, then you already account for the junction potential in your voltage protocols, resulting in nice round numbers.
There you go, and hope that helps. Questions? Let em rip in the comments.
