The overall goal of this procedure is to isolate F1 F zero APAs vesicles from rat brain for performing patch clamp recordings. This is accomplished by first removing the brain from a rat and homogenizing it. The second step is to disrupt synaptic zones by applying pressure.
Next, the synaptosome are layered onto the phial gradients. The final step is to incubate with digit toin and L to obtain the final vesicles for patch clamp recording. An important function of mitochondria is a TP production by oxidative phosphorylation.
The enzyme responsible for a TP production is the A TP synthase located at the mitochondrial inner membrane. To study the A TP synthase function in detail, we isolate special sub mitochondrial vesicles that we call SMVs. They contain the A TP synthase enzyme in an inside out configuration.
We then record these SMVs with a patch clamp electrode, and we determine the leakiness of their membrane. That tells us about the efficiency of a TP production by the mitochondria. Demonstrating this procedure today will be Sylvia ti, a postdoctoral fellow in my laboratory at Yale University.
In this procedure, after obtaining the head of a rat by decapitation, cut the skin to expose the skull, then open the skull gently by cutting it with a pair of scissors or Rons. Subsequently, remove the brain, mince the brain without the cerebellum finally in isolation, buffer, then transfer it to a five milliliter glass Teflon homogenizer gently homogenize the tissue 10 times, approximately five minutes in total. After that, centrifuge the sample at 1, 500 times G for 10 minutes at four degrees Celsius in a benchtop centrifuge.
Save the supernatant with mitochondria and synaptosome and discard the pellet. Then centrifuge it again at 16, 000 times, G for 10 minutes at four degrees Celsius in a benchtop centrifuge. Next, discard the supernatant and resuspend the palate In 500 microliters of isolation buffer disrupt the synaptosome with a cell disruption vessel by applying a pressure of 1, 200 PSI for 10 minutes, followed by rapid decompression layer the disrupted synap zones onto the centrifuge tubes with two different concentrations of fial that will form two layers.
Next place the tubes in an SW 50.1 rotor and centrifuge at 126, 500 times G for 20 minutes at four degrees Celsius. In an ultra centrifuge, the palate is the purified mitochondria. The layer between the different densities of fial is the undisrupted synaptosome.
Afterward, wash the pellet by centrifuging in isolation. Buffer at 16, 000 times G for 10 minutes at four degrees Celsius in a benchtop centrifuge. Now Resus, suspend the mitochondria in 200 microliters of isolation buffer, combined with an equal volume of 1%digit toin.
Place it on ice for 15 minutes. Next, add more isolation, buffer and centrifuge at 16, 000 times G for 10 minutes at four degrees Celsius. Repeat this procedure twice, then resuspend the palate in 200 microliters of isolation buffer, and add two microliters of 10%Lu bra, PX, mix and place on ice for 15 minutes.
After that, layer the mixture in isolation, buffer in the centrifuge tubes. Place the tubes in an SSW 50.1 rotor and centrifuge it at 182, 000 times G at four degrees Celsius for one hour. After an hour.
Wash the final palette by centrifuging it in isolation buffer at 16, 000 times G for 10 minutes at four degrees Celsius. A typical electrophysiology rig includes an amplifier, a PC computer equipped with a digi data 1, 440, a analog to digital converter interface in conjunction with P clamp, 10.0 software manipulators a microscope, a vibration isolation table, and a faraday cage. First, pull a pipette from a Boros silicate glass capillary tube in a flaming brown micro pipette puller.
The optimal resistance of the pipette should be between 80 and 100 mega ohm. Add the sub mitochondrial vesicles in a physiological intracellular solution. Then fill a patch clamp pipette with the same solution.
Visualize the vesicles under phase contrast. Microscopy after SMVs are patched at room temperature. Record the activity when the membrane potential is maintained at the voltage ranging from negative 100 millivolts to positive 100 millivolts for 10 seconds each period.
The data recorded is filtered at five kilohertz using the amplifier circuitry and analyzed with clamp fit 10.0 software. This figure shows the blot of lysate prepared from cytosol and mitochondria. The upper panel shows an immuno blot using an antibody against the cytosolic protein GA dh.
The bottom panel shows an blot using an antibody against the mitochondrial inner membrane protein. Cox four Here are two representative patch clamp recordings before and after the addition of one millimolar a TP.The holding potential is positive 70 millivolts. The dashed line represents zero picoamps.
Each trace is recorded for 10 seconds and there are 10 seconds between traces and shown. Here are the example traces of the fluorescence intensity changes of the A CMA indicator over time in the presence of SMVs and in the absence and presence of a TP.After watching this video, you should have a good understanding of how to isolate mitochondria, a sub mitochondria vesicle to containing F1 FO 80 TP Synthes. Moreover, you should understand out form I resistant seal with an electrode on the membrane of the vesicle to analyze the channel activity of the A TP Syntase Complex.
