Overview of HEPES Medium
HEPES medium is used to stabilize pH in a range of biological applications. Unlike bicarbonate buffers that rely on CO2, HEPES buffer operates independently of CO2 concentration, making it useful for experiments outside CO2 incubators.
Key Features
- pH Stability: HEPES effectively buffers in the pH range of 6.8 to 8.2, with an optimal buffering capacity at pH 7.2 to 7.4.
- Minimal Interference: It has low ionic strength and does not interfere significantly with biological processes or assays.
- Temperature Independence: HEPES maintains its buffering capacity over a wide temperature range, which is advantageous in varied experimental conditions.
Applications
- Cell Culture: HEPES is used in cell culture media to maintain pH in the physiological range, especially in experiments conducted outside CO2 incubators.
- Enzyme Assays: It provides stable pH conditions essential for accurate enzyme activity measurements.
- Protein Purification: HEPES helps maintain the stability and activity of proteins during purification processes.
- Electrophoresis: Used as a buffer in electrophoretic techniques to separate biomolecules.
Preparation
HEPES medium is typically prepared by dissolving HEPES in distilled water, adjusting the pH with NaOH or HCl, and then sterilizing the solution by filtration. It can be used at concentrations ranging from 10 to 50 mM, depending on the specific requirements of the experiment.
Limitations
- Light Sensitivity: HEPES can degrade under prolonged exposure to light, potentially generating toxic by-products.
- Cost: It is relatively more expensive compared to other common buffers like phosphate-buffered saline (PBS).
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