Adding HEPES to DMEM

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heehawmcduff's picture
Adding HEPES to DMEM

Can anyone provide any advice as to why someone would be adding HEPES to DMEM for general cell culture?  It was my impression that DMEM was a pretty good buffer in itself (no pun intended).

Does it have any significance if the person is intending to do a cell based ELISA downstream of any treatments?

Sami Tuomivaara
Sami Tuomivaara's picture


HEPES adds extra pH stability to your medium. DMEM is buffered mostly by the bicarbonate buffer and needs carbon dioxide from the atmosphere to maintain the proper equilibrium between bicarbonate and carbonic acid, and hence pH. Cell metabolism and taking your plates out of the incubator to ambient CO2 concentration can change the pH more than the bicarbonate can handle.


g a
g a's picture
HEPES is a componenet of Good

HEPES is a componenet of Good's buffer and is one of the strongest buffers in buffering range of 7.2 to 7.6 which also falls inn the physiological pH range.
HEPES is better at maintaining physiological pH despite changes in carbon dioxide concentration internally produced as a biproduct of cellular respiration The dissociation of water decreases with decrease in temperature, but the dissociation constants (pK) of many other buffers do not change much with temperature. However HEPES acts similar to water in that its dissociation decreases as the temperature decreases. This makes HEPES a more effective buffering agent for maintaining enzyme structure and function at low temperature.