HEPES

HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ) is a zwitterionic organic chemical buffering

agent; one of the twelve Good's buffers. HEPES is widely used in cell culture, largely because it is better
at maintaining physiological pH despite changes in carbon dioxide concentration (produced by cellular
respiration) when compared to bicarbonate buffers, which are also commonly used in cell culture. The
dissociation of water decreases with falling temperature, but the dissociation constants (pK) of many
other buffers do not change much with temperature. HEPES is like 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 temperatures.[1] Lepe-Zuniga et al. reported a
phototoxicity of HEPES when exposed to ambient light by the production of hydrogen peroxide[2][3],
which is not a problem in bicarbonate-based cell culture buffers. It is therefore strongly advised to keep
HEPES-containing solutions in darkness as much as possible. Fears that HEPES may serve as a nutrient
source for aerobic bacteria have been shown to be unfounded.

DIETHYLPYROCARBONATE (DEPC)

Diethylpyrocarbonate (DEPC), also called diethyl dicarbonate (IUPAC name), diethyl

oxydiformate, ethoxyformic anhydride, or pyrocarbonic acid diethyl ester, is used in the
laboratory to inactivate the RNase enzymes from water and other laboratory utensils. It
inactivates the RNases by the covalent modifications of the histidine residues. DEPC cannot be
used with Tris buffer or HEPES since they inactivate DEPC by reacting with it. In contrast it can
be used with PBS or MOPS. A handy rule is that enzymes or chemicals which have active -O:, -
N: or -S: cannot be treated with DEPC to become RNase-free as DEPC reacts with these species.
Furthermore DEPC degradation products can inhibit in vitro transcription.

Water is usually treated with 0.1% v/v diethylpyrocarbonate for at least 1 hour at 37°C and then
autoclaved (at least 15 min) to inactivate traces of DEPC. Inactivation of DEPC in this manner
yields CO2, H2O and EtOH. Higher concentrations of DEPC are competent of deactivating larger
amounts of RNase but remaining traces or byproducts may inhibit further biochemical reactions
such as in vitro translation. Further on, chemical modification of RNA such as
carboxymethylation is possible when traces of DEPC or its byproducts are present, resulting to
reduced usage of RNA even after buffer exchange (after precipitation).

DEPC treated water for use in a laboratory

DEPC treated (and therefore RNase-free) water is used in handling of RNA in the laboratory, to
reduce the risk of RNA being degraded by RNases.

DEPC derivatization of histidines is also used to study the importance of histidyl residues in
enzymes. Modification of histidine by DEPC results in carbethoxylate derivates at the N-omega-
2 nitrogen of the imidazole ring. DEPC modification of histidines can be reversed by treatment
with 0.5M hydroxylamine at neutral pH.
MOPS

MOPS is the common name for the compound 3-(N-morpholino)propanesulfonic acid, a

buffer introduced by Good et al. in the 1960s. It is a structural analog to MES.[1] Its chemical
structure contains a morpholine ring. HEPES is a similar pH buffering compound that contains a
piperazine ring. With a pKa of 7.20, MOPS is an excellent buffer for many biological systems at
near-neutral pH.

CHES

N-Cyclohexyl-2-aminoethanesulfonic acid, also known as CHES, is a buffering agent.

Typically appears as a white crystalline powder.

Decomposition or burning may produce toxic fumes such as carbon monoxide, carbon dioxide,
nitrogen oxides and sulfur oxides.

CHES buffers have a useful range of pH 8.6–10.

HEPPS

HEPPS or EPPS are the common names for the compound 3-[4-(2-Hydroxyethyl)-1-
piperazinyl]propanesulfonic acid. It is used as a buffering agent in biology and biochemistry. Its
chemical structure contains a piperazine ring. The pKa of HEPPS is 8.00.

MES

MES is the common name for the compound 2-(N-morpholino)ethanesulfonic acid. Its chemical
structure contains a morpholine ring. It has a molecular weight of 195.2 and the chemical formula is
C6H13NO4S. Synonyms include: 2-morpholinoethanesulfonic acid; 2-(4-morpholino)ethanesulfonic acid;
2-(N-morpholino)ethanesulfonic acid; 2-(4-morpholino)ethanesulfonic acid; MES; MES hydrate; and
morpholine-4-ethanesulfonic acid hydrate. MOPS is a similar pH buffering compound which contains a
propanesulfonic moiety instead of an ethanesulfonic one.