Choosing a biological buffer: HEPES, Tris, MES, MOPS and PIPES compared

A buffer only works well within about one pH unit of its pKa. Pick the wrong one and your pH drifts with temperature, your metal ions precipitate, or your assay reads high. Here is how the five most common research buffers compare, and when to use each.

Start with the working pH

The single most useful rule: choose a buffer whose pKa sits close to the pH you need at the temperature you will work at. The Good's buffers (MES, PIPES, MOPS, HEPES) were designed for biology. They are zwitterionic, poorly membrane-permeant and bind most metal ions weakly. Tris is older and cheaper, and still the default for many molecular-biology protocols.

Watch the temperature coefficient

Tris is the buffer most people get caught out by. Its pKa falls by roughly 0.03 units per °C, so a Tris buffer set to pH 7.5 at room temperature reads close to pH 8.0 on ice and nearer 7.2 at 37 °C. Always adjust the pH at the temperature you will use it. HEPES and MOPS drift about half as much, which is one reason HEPES is preferred for incubator work.

Know the interferences

• Tris carries a primary amine. It reacts with aldehydes and NHS esters, so avoid it in crosslinking and amine-coupling steps. It can also interfere with some protein assays and with certain pH electrodes.
• HEPES can generate hydrogen peroxide when exposed to light in the presence of riboflavin, which matters for long cell-culture exposures. Store media containing HEPES in the dark.
• Phosphate (Potassium phosphate, monobasic) is cheap and well understood, but precipitates with calcium and magnesium and inhibits some kinases and phosphatases.
• PIPES free acid is poorly soluble in water. Suspend it and add NaOH or KOH until it dissolves, then fine-tune the pH.
• MES contains no amine or carboxyl group, which makes it the standard choice for EDC/NHS activation steps.

Pick the right grade

Most of these buffers are listed in several grades. For routine buffers, Extra Pure / LR is fine. For nucleic-acid work, choose a molecular biology grade, which is tested for nuclease activity. For media preparation, choose cell culture tested material. Our Molecular Biology Grade and Buffer Preparation collections make this easier to filter. For ready-to-dilute buffers, Tris-EDTA 100X solution saves a step in DNA storage and elution workflows.

Products in this guide

HEPESCAS 7365-45-9 Tris baseCAS 77-86-1 Tris hydrochlorideCAS 1185-53-1 MES free acidCAS 4432-31-9 MOPS hemisodium saltCAS 117961-20-3 PIPESCAS 5625-37-6 Potassium phosphate, monobasicCAS 7778-77-0 Tris-EDTA 100X solutionCAS 38641-82-6

Related collections

Buffer Preparation 106 Buffers & Detergents 90 Core Biochemicals 342 Molecular Biology Grade 136