Dialysis & buffer exchange
A pre-assembled cassette or a length of tubing holds the sample behind a membrane of known cut-off, immersed in stirred buffer. Slow but gentle, and the only format that reaches micromolar residues; needs several buffer changes, and the sample gains 10–20% volume by osmosis. Hydrate the membrane before loading, leave headroom, stir the buffer, and make the outer buffer the one the protein should live in.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Dialysis cassettes
A pre-assembled cassette or a length of tubing holds the sample behind a membrane of known cut-off, immersed in stirred buffer. Slow but gentle, and the only format that reaches micromolar residues; needs several buffer changes, and the sample gains 10–20% volume by osmosis. Hydrate the membrane before loading, leave headroom, stir the buffer, and make the outer buffer the one the protein should live in.
How it differs from the standard method
- Cassettes are easier and less lossy than knotted tubing, but tubing is cheaper and comes in more cut-offs and volumes.
- Tubing often needs pre-treatment (boiling in EDTA/bicarbonate) to remove glycerol, sulfides and trace metals before use with sensitive samples.
- Equilibration time scales with sample volume and inversely with membrane area, so a thin cassette exchanges faster than a fat sausage of tubing.
- Two short changes followed by an overnight change is the usual compromise between the volume-ratio floor and the time each change needs.
1A membrane that sorts by size, and a gradient that does the work
Buffer exchange separates a macromolecule you want to keep from the small molecules around it (salt, imidazole, urea, free dye, nucleotides, reducing agent) using a membrane with pores small enough to hold the macromolecule back and large enough to let the small molecules through. Nothing pushes them out: they diffuse down their own concentration gradient until the concentration inside the membrane matches the concentration outside. That single fact determines everything about how dialysis is planned.
Volume ratio sets the floor
R is buffer volume over sample volume. One change cannot go below 1/(1+R), however long you leave it.
Changes multiply
Each fresh change applies the floor again. Two short changes plus an overnight beat one very long one.
Time gets you to the floor
Each change approaches its own equilibrium exponentially. A 2 h change typically covers only about two-thirds of the way.
2The cut-off is a nominal figure, not a wall
A membrane's molecular weight cut-off is the mass at which it retains some stated fraction, often 90%, of a test solute, usually globular proteins or dextrans. It is a label on a distribution of pore sizes, not a sieve with one hole size.
3Three ways to do the same job
- Dialysis
- Slowest and gentlest, and the only one that reaches micromolar residues. Hours to overnight, needs several changes, and the sample is diluted 10–20% by osmotic water influx.
- Desalting column
- Minutes. Size exclusion holds the small molecule up in the beads while the protein passes through the void volume. Removes 95–99% of the salt, not 99.99%, and always dilutes the sample into the void volume.
- Spin concentrator
- Concentrates and exchanges at once, but only by repetition: salt passes the membrane freely, so its concentration in the retentate does not change until you dilute back and re-concentrate.
4Where the sample goes
Losses in buffer exchange are rarely mysterious, and the measurement that tells you which one happened costs one A280 reading on the other side of the membrane. Protein passing the membrane shows up in the dialysate or the filtrate.
5Keeping the protein alive while the buffer changes under it
The protein does not get to choose when the conditions change. Over a dialysis the ionic strength, the pH and the concentration of every stabiliser fall towards whatever is in the outer buffer, so the outer buffer is the buffer the protein will live in, and it has to be one the protein is stable in.
6Concentration polarisation and fouling
A spin concentrator forces buffer through the membrane, which drags protein onto it faster than diffusion can carry it away. The layer against the membrane becomes far more concentrated than the bulk retentate, and that layer resists flow: flux falls steeply as the spin goes on, so the last few hundred microlitres take as long as the first several millilitres.
Key terms
Practise these as flashcards →Molecular weight cut-off: the mass at which a membrane retains a stated fraction (often 90%) of a test solute. A nominal label on a pore-size distribution.
Buffer volume divided by sample volume. One change can at best leave 1/(1+ratio) of the solute behind.
Exchanging a buffer in a concentrator by repeatedly diluting the retentate with new buffer and re-concentrating. Each cycle divides the permeating solute by the concentration factor.
The liquid outside the beads of a size-exclusion bed. Fully excluded molecules elute in it; this is also what dilutes a desalted sample.
The concentrated layer of retained solute that builds against an ultrafiltration membrane under flow, reducing flux and trapping protein.
The ratio of a solute's concentration in the permeate to its concentration in the retentate. Zero is perfect retention; one is free passage.
The quiescent film of liquid against a membrane. It makes the local gradient much shallower than the bulk gradient, which is why dialysis buffer is stirred.
When to use it
- Taking a protein out of an elution buffer that would interfere downstream: imidazole before ion exchange, high salt before loading a column, glutathione or biotin after affinity elution.
- Removing a denaturant or a reducing agent slowly, which is how refolding from urea or guanidine is usually attempted.
- Getting rid of free label after a conjugation, or free nucleotide after a kinase reaction, where a desalting column is fast enough.
- Concentrating a dilute pool for crystallography, a binding experiment or storage, where only a spin concentrator does both jobs at once.
- Matching a sample and a reference buffer exactly, as calorimetry and circular dichroism require.
Compared with alternatives
A full size-exclusion run separates by size with resolution; a desalting column is the same principle run coarsely for speed.
Precipitation also changes the solution around a macromolecule, and far faster, but it means taking the sample out of solution and back again.
What drives a spin concentrator, and what you use to clear the aggregate that a buffer change leaves behind.
The measurement that tells you what the exchange cost, and whose own reagents are sensitive to the buffer you have just moved into.
Also a membrane process, but separating particles from liquid rather than small solutes from macromolecules.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
A pre-assembled cassette or a length of tubing holds the sample behind a membrane of known cut-off, immersed in stirred buffer. Slow but gentle, and the only format that reaches micromolar residues; needs several buffer changes, and the sample gains 10–20% volume by osmosis. Hydrate the membrane before loading, leave headroom, stir the buffer, and make the outer buffer the one the protein should live in.
- Cassettes are easier and less lossy than knotted tubing, but tubing is cheaper and comes in more cut-offs and volumes.
- Tubing often needs pre-treatment (boiling in EDTA/bicarbonate) to remove glycerol, sulfides and trace metals before use with sensitive samples.
- Equilibration time scales with sample volume and inversely with membrane area, so a thin cassette exchanges faster than a fat sausage of tubing.
- Two short changes followed by an overnight change is the usual compromise between the volume-ratio floor and the time each change needs.