pH of clearing solution

Does anyone know the reason behind the clearing solution having to be at a pH of 8.5?

Comments

  • JAGSJAGS Posts: 16
    I don't know really, but I think that it's a nice pH to SDS reaction and the sample, due the [protons]
  • For the micelles:

    CLARITY wiki, section 4.2 in Protocol >> ETC
    http://wiki.claritytechniques.org/index.php/Electrophoretic_Tissue_Clearing
    "Check pH routinely
    Acid accumulation in the clearing solution could damage the tissue sample and also disrupt the structure of the ionic micelles, thereby negating their ability to clear lipids from the tissue. The presence of boric acid in the clearing solution helps to buffer the acid build-up, but it can only slow, and not counteract, its effect on the pH of the circulating clearing solution. To keep the SDS micelles intact, it is important to keep the pH level above 7. It is recommended to regularly check the clearing solution pH (once or twice a day) and change the clearing solution when the pH dips into the 7.5-8 range. Always use fresh clearing solution when replacing the circulating buffer (do not rebalance the pH in the old buffer using sodium hydroxide).
    Some variables in the ETC set-up and settings may increase clearing solution degradation due to acid accumulation, thereby necessitating replacement of the clearing solution more often. Such variables include higher voltage settings (40-50 V) and multiple chambers connected in parallel to one another (same volume of clearing solution is exposed to more electrophoresis in total than with only one chamber).
    Lowering the voltage setting applied to the ETC chamber will decrease the rate of clearing solution degradation. If the ETC chamber is running at a low enough voltage such that acid accumulation does not show significant effects on the pH, it is recommended to replace the clearing solution after every 1-2 samples anyways. Clearing is always more efficient with a fresh batch of clearing solution."
  • Does the yellow colour of the clearing buffer can be used as a sign of degradation? Thanks
  • FabioFabio Posts: 61
    Hi I never seen the clearing buffer turn yellow. I would recommend changing the buffer daily
  • LeoLeo Posts: 9
    Hi, I'v never seen yellow color of buffer. I'm not making buffer by myself, just purchasing commertial one. Moreover, I think it's not a main problem with pH from removed lipid acid. The key of important thing may be from broken SDS.
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