pH Shift over ETC

AReady

What kind of pH shifts has everyone been seeing over the course of their ETC? At 30V/40C or higher my pH has been dropping below 7 within 3 or so days, which has necessitated switching to a fresh batch of clearing solution after every tissue sample. Since we're planning to scale up to a few chambers in parallel, I wanted to see if anyone has similar experiences/suggestions about pH.

joelrosiene

I too have been swapping out my clearing fluid every 3 or so days for the same reasons. On one run, instead of swapping out the solution, I just gave it a few squirts of NaOH back up to pH 8.5 and I haven't seen any ill effects. Anyone have thoughts on just adding more NaOH to fix the acidification?

Kristin_Engberg

Adding more NaOH to readjust the pH of the clearing buffer will alter the ion concentration within the solution. This will change the formation and structure of the SDS micelles and could lead to significantly reduced clearing. I would recommend replacing the solution with fresh buffer as you have been doing. You may be able to make your clearing buffer last longer by adjusting your power and flow rate settings.

Suzschindler

When I use 1.6 L of solution (bottle in a water bath instead of full water bath), my pH drops from 8.5 to 8.3 or 8.2 in 24 hours. How acidic can the buffer get before the sample is damaged?

joelrosiene

@Suzschindler I'm not sure about damage, but I change it whenever it hits 7 or so. I wouldn't go below that.

nparker327

I tracked my latest brain as it ran through ETC. At 0 hours pH= 8.42. 24hours = 8.3. 48 hours = 7.83. 72 hours = 7.59. 96 hours = 7.4 (32V, 36 degrees)

nparker327

I have heard that 7.5 is the most acidic the clearing solution should be.