Talk:Cleaning the TPP+ electrodes
Question:I have some troubles using the TPP-electrodes. The problem consist out of 2 parts. First, I don't seem able to get rid of all the inhibitors after an experiment. After the standard cleaning procedure, I let the electrodes stand in distilled water for about 20 minutes and then I put them into a homogenate for about 20 minutes as well. Still, in the next experiment, respiration is inhibited. When removing the electrodes, rinsing the cells and doing standard measurement (without TPP electrodes inserted), respiration is just fine. The only time I get a good measurement is after I replaced the membranes of the TPP-electrodes. This is impossible to do for every experiment. Can you give me any advice on this part?
Second, I also suspect biological contamination of one of my TPP electrodes. Oxygen flux in the closed chamber, without biological material, is too high (~10pmol/(s*ml)). This isn't the case when measuring without the TPP electrodes. So, I'm quite sure that the contamination must come from the TPP or reference electrode. How can I get rid of this? I didn't want to take the risk to put the electrodes in 70% EtOH because I'm not sure if this would be ok? What is your idea on this?
Answer 1: I guess the standard cleaning procedure mentioned already included rinsing with ethanol? If the cleaning with biological materiel does not work the third option of cleaning is to really immerse then in ethanol, see http://www.oroboros.at/fileadmin/user_upload/O2k-Manual/MiPNet15.03_MultiSensor-ISE.pdf This will however, decrease the lifetime of the electrode. Since you seem to be in the luck position to have homogenate available for washing I would not immediately implement the immersion in ethanol as standard procedure. You write that you put the in homogenate: is this outside the O2k? If yes would put the homogenate into the O2k chambers activate stirring and have the temperature at 37 degree for this homogenate washing procedure.
The second question is related to the first. You rinse the TPP electrode with ethanol anyway. You can immerse it in ethanol, but this will degrade membrane lifetime. But since the other option is to disassemble the electrode anyway you should try the immersion in 70 % ethanol. If this does not help and you have to disassemble the electrode: wash all the individual components of the electrode first with water and then with 100% or 70% ethanol for indefinitely long times. If you saw biolgicl contamination for a TPP electrtode rmember to also throw away the storage solution of the electrode. For the reference electrode the problem with long exposure to Ethanol is potential clogging of the fritt. If you suspect biological contamination I would just rotate the glass barrel: Use a new glass barrel for the time being. In the meanwhile clean the old barrel first with water (soak it fin water) to red rid of the salts) the you can immerse it in 70% Ethanol as long as you want to. Before refilling the glass barrel clean it again with a lot of water to get rid of all the ethanol.--Fasching Mario 13:24, 3 May 2012 (CEST)
Answer 2: 1. Unfortunately, I had the same problem with inhibitors - sometimes I could not get rid of it by the procedure you just explained. Maybe the most effective was inserting electrodes into the oxygraph chamber with homogenate like in experiment. You have continuous stirring and 37°C... But even this did not give 100% success. 2. Contamination of the TPP electrode can also be a problem. It is necessary time to time exchange the storage solution and keep electrodes in the darkness. I could see several times something growing inside the storage solution, but never had such a high O2 flux after good rinsing and keeping in fresh solution. You can easily clean the reference electrode by removing the liquid, cleaning the glass part and putting it into ethanol. It is easy and fast. For cleaning the TPP electrode you would need to remove the membrane, clean everything with ethanol, condition the electrode... it would take 3 days.
So - maybe the only solution for standard experiments would be to choose a different approach - choose some particular problem to solve: the effect of stepwise addition of ATP on membrane potential, the effect of Ca2+...on mitochondria of control and affected animals...
Maybe malonic acid does not stick to the membrane of TPP electrode - it is successful used in the literature, see this paper:
Borutaite V, Morkuniene R, Budriunaite A, Krasauskaite D, Ryselis S, Toleikis A, Brown GC. Kinetic analysis of changes in activity of heart mitochondrial oxidative phosphorylation system induced by ischemia. J Mol Cell Cardiol. 1996 Oct;28(10):2195-201. PubMed PMID: 8930814.
I know from Vilma, that they keep the electrode contaminated with rotenone only for measurements with rotenone. And they have long-lasting experience with home-made TPP electrodes.
So - my advice is to read the literature and find a good application for TPP electrode, where you don´t need to use lipid-soluble inhibitors... It is also my task now, but I did not get to it yet.
