Affinity purification
His₆–₁₀ tags bind Ni²⁺ or Co²⁺ resin. Cheap, high capacity, works under denaturing conditions; moderate specificity, so imidazole washes and a polishing step are usual.
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His-tag / IMAC
His₆–₁₀ tags bind Ni²⁺ or Co²⁺ resin. Cheap, high capacity, works under denaturing conditions; moderate specificity, so imidazole washes and a polishing step are usual.
How it differs from the standard method
- EDTA-free inhibitors.
- ≤ 5 mM β-ME or TCEP; avoid DTT unless the resin tolerates it.
- Co²⁺ (TALON) gives higher purity, lower yield than Ni²⁺.
1Fishing one protein out of a lysate
Affinity purification uses a tag fused to your protein and a resin carrying something that binds the tag tightly and specifically. The lysate flows over the resin, the tagged protein sticks, other proteins wash away, and a competitor or a change in conditions releases the pure protein. One step can take a protein from a fraction of a percent of the lysate to over 90% purity.
His-tag (IMAC)
Six histidines coordinate nickel on NTA resin. Imidazole, which looks like histidine's side chain, competes them off.
GST tag
Glutathione S-transferase binds glutathione resin; free glutathione elutes it.
Strep-tag II
An 8-residue peptide binds engineered streptavidin; desthiobiotin elutes gently.
Protein A/G
Bacterial proteins that bind antibodies' Fc; low pH elutes them.
2Bind, wash, elute
Every affinity step has the same logic: binding conditions that favour the tag–resin interaction, wash conditions just stringent enough to remove weakly bound host proteins, and elution conditions that release the tagged protein. In IMAC, 20–40 mM imidazole in the wash removes most E. coli proteins with histidine patches, and 250–500 mM elutes the His-tagged protein. Chelators (EDTA) and strong reducing agents (DTT) strip or reduce the nickel and must be kept out.
3Reading the fractions
Run the lysate, flow-through, washes and elution fractions side by side on an SDS-PAGE gel. Your protein in the flow-through means it didn't bind (resin full, nickel stripped, tag hidden); in the wash means the wash was too stringent; still faint in the elution means it didn't come off.
Key terms
Practise these as flashcards →A peptide or protein fused to the target to give it a handle for purification.
Immobilised metal affinity chromatography: His-tags bind metal ions held by a chelating resin.
Milligrams of tagged protein a millilitre of resin can hold.
What passes the column without binding.
Histidine-like competitor used in IMAC wash (low) and elution (high) buffers.
Further purification steps (SEC, ion exchange) after the affinity capture.
When to use it
- Purifying recombinant proteins for structural, biochemical or antibody work.
- Pulling down a tagged bait together with its binding partners (with gentler washes).
- Purifying antibodies from serum or hybridoma supernatant (Protein A/G).
Compared with alternatives
Size exclusion and ion exchange separate by size and charge after the affinity capture.
Where the tagged protein comes from.
The standard way to judge purity of the fractions.
The same chemistry used to find interaction partners rather than to purify.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
His₆–₁₀ tags bind Ni²⁺ or Co²⁺ resin. Cheap, high capacity, works under denaturing conditions; moderate specificity, so imidazole washes and a polishing step are usual.
- EDTA-free inhibitors.
- ≤ 5 mM β-ME or TCEP; avoid DTT unless the resin tolerates it.
- Co²⁺ (TALON) gives higher purity, lower yield than Ni²⁺.