PH calibration buffers: Difference between revisions
From Bioblast
No edit summary |
No edit summary |
||
Line 2: | Line 2: | ||
|description='''pH calibration buffers''' are prepared to obtain two or more defined pH values for calibration of pH electrodes and pH indicator dyes. | |description='''pH calibration buffers''' are prepared to obtain two or more defined pH values for calibration of pH electrodes and pH indicator dyes. | ||
}} | }} | ||
{{MitoPedia methods | {{MitoPedia methods | ||
|mitopedia method=Respirometry, Fluorometry | |mitopedia method=Respirometry, Fluorometry | ||
}} | }} | ||
{{MitoPedia topics | {{MitoPedia topics | ||
|mitopedia topic=Medium | |mitopedia topic=Medium | ||
}} | }} | ||
== | == Modified hanks buffer == | ||
{| class="wikitable" border="1" | |||
|- | |||
! Substance | |||
! Formula | |||
! Concentration [mM] | |||
|- | |||
| Calcium chloride dihydrate | |||
| CaCl<sub>2</sub> * 2 H<sub>2</sub>O | |||
| 0.42 | |||
|- | |||
| Imidazole | |||
| C<sub>3</sub>H<sub>4</sub>N<sub>2</sub> | |||
| 2.0 | |||
|- | |||
| Potassium chloride | |||
| KCl | |||
| 2.4 | |||
|- | |||
| Magnesium chloride hexahydrate | |||
| MgCl<sub>2</sub>*6 H<sub>2</sub>O | |||
| 0.49 | |||
|- | |||
| Sodium chloride | |||
| NaCl | |||
| 137 | |||
|} | |||
::::* pH adjusted to 7.1 at 30 °C. | |||
::::* pH adjusted with KOH or HCl to create a buffer series: (pH values at 25 °C) | |||
::::# A (pH 3.3), | |||
::::# B (pH 6.1) | |||
::::# C (pH 6.6) | |||
::::# D (pH 6.9) | |||
::::# E (pH 7.3) | |||
::::# F (pH 7.5) | |||
::::# G (pH 11.1) pH 10.1 at 37° did not represent the basic endpoint for carboxy-SNARF-1. | |||
:::'''Calibration''' | |||
::::* Basic endpoint (for SNARF): fluorophore completely deprotonated. | |||
::::* Acidic endpoint (for SNARF): fluorophore completly protonated. | |||
== Media with low buffering capacity == | |||
:::: Media with low pH buffering capacity are required for measurement of proton flux in intact cells. Commercially available variants of RPMI1640 that contain neither hydrogen carbonate nor HEPES may constitute a starting point, see e.g. Sigma Product number 6504 (not tested by Oroboros Instruments, due to the inclusion of phenol red this medium may (or may not) be problematic for fluorescence). |
Revision as of 10:46, 8 September 2018
Description
pH calibration buffers are prepared to obtain two or more defined pH values for calibration of pH electrodes and pH indicator dyes.
MitoPedia methods:
Respirometry,
Fluorometry
MitoPedia topics:
Media for respirometry
Modified hanks buffer
Substance | Formula | Concentration [mM] |
---|---|---|
Calcium chloride dihydrate | CaCl2 * 2 H2O | 0.42 |
Imidazole | C3H4N2 | 2.0 |
Potassium chloride | KCl | 2.4 |
Magnesium chloride hexahydrate | MgCl2*6 H2O | 0.49 |
Sodium chloride | NaCl | 137 |
- pH adjusted to 7.1 at 30 °C.
- pH adjusted with KOH or HCl to create a buffer series: (pH values at 25 °C)
- A (pH 3.3),
- B (pH 6.1)
- C (pH 6.6)
- D (pH 6.9)
- E (pH 7.3)
- F (pH 7.5)
- G (pH 11.1) pH 10.1 at 37° did not represent the basic endpoint for carboxy-SNARF-1.
- Calibration
- Basic endpoint (for SNARF): fluorophore completely deprotonated.
- Acidic endpoint (for SNARF): fluorophore completly protonated.
- Calibration
Media with low buffering capacity
- Media with low pH buffering capacity are required for measurement of proton flux in intact cells. Commercially available variants of RPMI1640 that contain neither hydrogen carbonate nor HEPES may constitute a starting point, see e.g. Sigma Product number 6504 (not tested by Oroboros Instruments, due to the inclusion of phenol red this medium may (or may not) be problematic for fluorescence).