This method can help with investigation of membrane proteins such as transporters and channels. The information about substrate turnover rate loss comparing protein activity and specificity and the different physiological and pathological conditions. The main advantage of this technique is that it minimizes potential shift variation and chloride free buffer conditions.
And significantly increases precision of turnover rate determination due to more exact membrane potential measurements. Gather the elements for the fist steps. These include two micro-capillary pipette tips, a sharp blade and a sliding caliper.
For the electrode have silver wire, sand paper and a three molar potassium chloride solution at hand. In addition have ethanol and water for cleaning and a DC power supply. Proceed by working with the two micro-pipette tips.
Start with the tip that will contain the buffer. Place the second tip next to the first. Move the second tip, so, when inserted it's narrow part will extend at least five millimeters into the narrow part of the first.
Mark the position where the first buffer containing tip will be attached. Then use the caliper to measure the length of the micro-agar salt bridge electrode. Use the blade to cut the tip to the appropriate length.
Clean the cut surface with ethanol followed by water. Next, get a length of approximately eight centimeters of silver wire for the electrode. Clean the wire on wipes with ethanol followed by water.
After cleaning, use sand paper to smooth the surface on a one centimeter length at one end. This wire with its smoothed end is ready to be electro-chemically coated. Put the smoothed end into the potassium chloride solution with the other end connected to a DC power supply for coating.
When coated, disconnect the electrode and clean it with water. After drying it, determine the length that allows it to penetrate the micro capillary tip as deeply as possible. Cut the electrode from the uncoated side to the appropriate length.
Now, move on to prepare a salt solution with agarose. In a flask containing water dissolve potassium chloride and use a magnetic stirrer to aid mixing. After removing the stirrer add agarose to the flask.
Take the flask to a microwave and heat it to melt the agarose at about 100 degrees Celsius. Take it out to visually check that the agarose is dissolved completely. Once the agarose is dissolved pipette 10 microliters into the micro-capillary slowly to avoid air bubbles.
It is important to carefully pipette the agar salt solution into the micro capillary tip, especially at high agar concentrations. If not done properly air bubbles are easily produced in the tip and block electrical flow. After removing the tip from the pipette push the electrode into it.
Ensure the electrode penetrates the salt solution. When the electrode is at room temperature plug it in a reference electrode into an amplifier. Support the reference electrode so it can be lowered into a plastic container with one one milliliter of buffer.
Next, dip the salt bridge electrode into the solution. Apply a voltage and check that there is a current response before continuing. If the test is successful, disconnect the electrodes.
If necessary, store the salt bridge electrode by dipping it in a three molar potassium chloride solution. Next, prepare the buffer containing plastic tip. Take a micro capillary tip and identify a point two centimeters from the narrow part.
Use a heating wire to bend the tube 90 degrees at this position. With a sharp knife cut the tube five millimeters from the bent. Clean the area that was cut with ethanol followed by water.
Before proceeding, use a light microscope with a scale to measure the diameter of the hole at the tip. Next, move the pipette to be near a container of solvent and another of buffer. Pipette three microliters of the solvent into and out of the tip.
Next, fill the measurement tip with three microliters of buffer. Now, retrieve the salt bridge from the potassium chloride solution. Plug the measurement tip with the buffer onto the salt bridge electrode.
Connect the salt bridge electrode and the reference electrode to an amplifier. At this point, suspend the salt bridge electrode in the buffer solution with the reference electrode. A representation of the configuration for the experiment is in this schematic.
During the experiment, the membrane will form at the end of the buffer containing pipette. Collect data to find the membrane capacity. Do this by applying a triangular alternating voltage signal to create a rectangular alternating current response.
Next, find the conductance and voltage at zero current. Automate the application of a voltage ramp ranging from minus 50 to 50 millivolts and record the current. Fit a linear function to the data.
The slope is the conductance, the x-intercept is the voltage at zero current. When done remove the measurement tip from the salt bridge. Fill a new measurement tip with buffer, adjust it to different pH value to set up a proton gradient across the membrane.
Re-establish the experimental set up with the a new measurement tip and electrodes in order to repeat the measurements. Here are representative current voltage recordings in the presence of a pH gradient. And, in the absence of a pH gradient.
The lines represent a linear fit to the data. The shift in the x-axis intersection point for the different pH values is predicted by the Nernst equation. These data represent the shift in the membrane potential in time for a standard silver chloride electrode in white, and the micro-agar salt bridge electrode in black.
The agar salt bridge electrode was more stable with a maximum shift of less than five millivolts over 300 seconds of measurement. In this plot of the potential shift as a function of pH gradient the two electrodes are seen to behave significantly differently as the pH gradient increases. The proton turnover rate can be found and compared for the two electrode types.
Here are proton turnover numbers determined for mitochondrial uncoupling protein one UCP1 measured by the salt bridge electrode compared to a standard electrode. There is also data for similarly prepared UCP3. The rates seem more precise with the agar salt bridge electrode.
While attempting this procedure it's important to remember to ensure that the electrode penetrates the agar salt solution. The salt bridge must also dip into the buffer solution. Finally, ensure that the agar salt solution contains no air bubbles.
