Protein tags — affinity, epitope & solubility
A reference table of the common protein tags used in recombinant expression: affinity tags for purification, epitope tags for detection, and solubility / fusion partners for folding. Amino acid sequences are shown in MONOSPACE; large fusion proteins list see notes with their approximate mass and source instead of a full sequence.
| Tag | Type | AA sequence | Length | Mass (kDa) | Purification / detection | Cleavable by | Notes |
|---|---|---|---|---|---|---|---|
| His6 (6×His) | Affinity | HHHHHH | 6 | 0.84 | Ni-NTA / Co (TALON) IMAC resin; anti-His mAb | — | Most common purification tag. Binds immobilized Ni2+/Co2+; elute with imidazole or low pH. Tag is not itself protease-cleavable — cleavage depends on a separate site (e.g. TEV, thrombin) engineered between tag and target. Codons often CAT/CAC mix. |
| His8 (8×His) | Affinity | HHHHHHHH | 8 | 1.1 | Ni-NTA / Co IMAC resin; anti-His mAb | — | Higher-avidity variant of His6; tighter IMAC binding, useful for low-expression or membrane proteins. Otherwise same handling as His6. |
| FLAG | Affinity | DYKDDDDK | 8 | 1.01 | Anti-FLAG M1/M2/M5 mAb; anti-FLAG (M2) affinity gel; elute with FLAG peptide or low pH | Enterokinase (cleaves after DDDDK↓) | Sigma/Millipore trademark. Hydrophilic, often surface-exposed. Enterokinase cleaves at the C-terminal DDDDK, leaving native N-terminus when FLAG is N-terminal. M1 antibody requires free N-terminal Asp (Ca2+-dependent). |
| 3×FLAG | Affinity | DYKDHDGDYKDHDIDYKDDDDK | 22 | 2.73 | Anti-FLAG M2 mAb / M2 affinity gel; 3×FLAG peptide elution | Enterokinase (at terminal DDDDK↓) | Sigma trademark. Tandem repeat gives ~10× more sensitive detection than single FLAG. ⚠ Exact junction residues (DHDGD / DHDID) are from the Sigma 3×FLAG design; sequence above is the standard published form. |
| Strep-tag II | Affinity | WSHPQFEK | 8 | 1.06 | Strep-Tactin / Strep-Tactin XT resin; StrepMAB; gentle elution with desthiobiotin/biotin | — | IBA Lifesciences trademark. Kd ~1 µM to Strep-Tactin. Very mild, physiological elution → good for intact complexes. Twin-Strep-tag (two copies, WSHPQFEK...GGGSGGGSGGSA...WSHPQFEK) binds much tighter; use it for demanding preps. |
| S-tag | Affinity | KETAAAKFERQHMDS | 15 | 1.75 | S-protein resin / S-protein–HRP (RNase S system); quantitative S-Tag assay | — | Novagen/Merck. Derived from the S-peptide of RNase A; binds S-protein to reconstitute RNase activity, enabling sensitive quantitation. Not designed as a standalone cleavage site. |
| HA (hemagglutinin) | Epitope | YPYDVPDYA | 9 | 1.1 | Anti-HA mAb (12CA5, 3F10, HA.11); anti-HA agarose | — | From influenza hemagglutinin HA1 (residues ~98–106). Widely used for Western/IP/IF; well-tolerated at N- or C-terminus. |
| c-Myc | Epitope | EQKLISEEDL | 10 | 1.2 | Anti-Myc mAb (9E10); anti-Myc agarose | — | From human c-Myc (residues 410–419). Classic detection/IP tag; 9E10 is the standard antibody. Often combined with His6. |
| V5 | Epitope | GKPIPNPLLGLDST | 14 | 1.42 | Anti-V5 mAb; anti-V5 agarose | — | Derived from the P/V proteins of simian virus SV5 (paramyxovirus). Common in Invitrogen/Thermo vectors (e.g. pcDNA). Low background, good for mammalian expression. |
| T7-tag | Epitope | MASMTGGQQMG | 11 | 1.16 | Anti-T7 mAb; T7-Tag antibody agarose | — | From the N-terminus (leader) of T7 gene 10 capsid protein. Novagen/Merck pET-system detection tag. ⚠ Length varies by vector: an 11-residue form (MASMTGGQQMG) is standard; some report the first ~11–13 residues. |
| GST (glutathione S-transferase) | Solubility | see notes | 218 | 26 | Glutathione-Sepharose / GSH resin (affinity); anti-GST mAb; elute with reduced glutathione | Thrombin or PreScission/HRV-3C (site depends on vector, e.g. pGEX) | Schistosoma japonicum GST, ~26 kDa (~218 aa). Dual affinity + moderate solubility enhancer. Forms dimers — can be a drawback for oligomerization studies. Full sequence: UniProt P08515 / Addgene pGEX vectors. Cleavage site (LVPR↓GS thrombin or LEVLFQ↓GP 3C) is vector-specific. |
| MBP (maltose-binding protein) | Solubility | see notes | 396 | 42.5 | Amylose resin (affinity); anti-MBP mAb; elute with maltose | Factor Xa, TEV, or PreScission/3C (site depends on vector, e.g. pMAL) | E. coli MalE, ~42.5 kDa. Strong solubility enhancer (soluble in ~70–80% of targets) plus amylose affinity. Full sequence: UniProt P0AEX9 / NEB pMAL vectors. ⚠ length_aa ~366–396 depending on signal-peptide/linker variant used. |
| SUMO (Smt3) | Solubility | see notes | 98 | 11 | Typically paired with an N-terminal His6 for Ni-NTA capture; no intrinsic affinity resin | SUMO protease (Ulp1 / SENP), cleaves after C-terminal di-Gly | Yeast Smt3 (~11 kDa) is the common form (LifeSensors "Champion SUMO"). Enhances solubility/expression; Ulp1 recognizes tertiary structure and cleaves after the C-terminal Gly-Gly, leaving a NATIVE N-terminus (any residue except Pro). Full sequence: UniProt Q12306 (Smt3) / Addgene pET-SUMO. |
| NusA | Solubility | see notes | 495 | 55 | No intrinsic affinity resin — used with a co-tag (e.g. His6) for purification | TEV or other vector-defined protease site | E. coli transcription factor NusA (~55 kDa). Very effective solubility enhancer, especially for toxic/aggregation-prone targets, but large — high metabolic burden and reduced molar yield. Full sequence: UniProt P0AFF6 / Novagen pET-44 (NusA·Tag). |
| Thioredoxin (Trx / TrxA) | Solubility | see notes | 109 | 12 | No intrinsic affinity resin — pair with His6/His-patch (ThioFusion); anti-Trx available | Enterokinase / thrombin / TEV (vector-dependent) | E. coli TrxA (~11.7 kDa, 109 aa). Compact, highly soluble; best for small targets (<30 kDa) without disulfides in the reducing cytoplasm. Full sequence: UniProt P0AA25 / Invitrogen pTrxFus, pET-32 (Trx·Tag). |
| HaloTag | Self-labeling | see notes | 297 | 33 | HaloTag ligands (chloroalkane): fluorophores, biotin, HaloLink resin — COVALENT capture | TEV (in Promega HaloTag vectors, a TEV site flanks the tag) | Promega. Engineered haloalkane dehalogenase (~33 kDa, ~297 aa) that forms an IRREVERSIBLE covalent bond to chloroalkane ligands → very stable pulldowns/labeling and one-step covalent immobilization. ⚠ mass cited variously as 33–34 kDa. Not a classic epitope/affinity peptide. |
| SNAP-tag | Self-labeling | see notes | 182 | 20 | O6-benzylguanine (BG) ligands: fluorophores, biotin, resin — COVALENT self-labeling | Vector-dependent (TEV/3C sites offered in some constructs) | NEB. Engineered human O6-alkylguanine-DNA alkyltransferase (hAGT, ~20 kDa, ~182 aa) that covalently reacts with benzylguanine substrates. CLIP-tag is a companion that reacts with benzylcytosine (orthogonal labeling). ⚠ mass ~19.4–20 kDa depending on construct. |
Choosing a tag
Start with the smallest tag that solves your problem. A His6 tag is the standard workhorse for purification; add an epitope tag like HA, c-Myc or V5 when you need antibody-based detection. If the target is insoluble or aggregation-prone, reach for a solubility partner (MBP, SUMO, NusA or Trx) — but remember these are large and reduce molar yield. Once purified, check the predicted mass and pI of your tagged construct with the protein molecular weight and pI guide.
Removing a tag
Most peptide tags do not cleave themselves — cleavage depends on a separate protease site engineered between the tag and your protein. Common choices are TEV, thrombin, PreScission/HRV-3C, enterokinase (for FLAG) and SUMO protease (which leaves a native N-terminus). The Cleavable by column above lists the protease each vector typically pairs with.
Frequently asked questions
- What is a protein tag?
- A protein tag is a short peptide or a whole fusion protein genetically added to your target's N- or C-terminus. Tags let you purify (affinity tags), detect (epitope tags), or improve the folding and solubility (solubility tags) of a recombinant protein.
- Which protein tag should I use for purification?
- His6 (6×His) is the default first choice: small, cheap Ni-NTA/Co IMAC purification that works under native or denaturing conditions. Use FLAG or Strep-tag II when you need very mild, specific elution (e.g. for intact complexes), and GST or MBP when the target also needs a solubility boost.
- What is the difference between an affinity tag and an epitope tag?
- Affinity tags (His6, Strep-tag II, GST, MBP) bind a resin or ligand so you can capture and elute the protein. Epitope tags (HA, c-Myc, V5, T7) are recognized by well-characterized antibodies and are used mainly for Western blot, immunoprecipitation and immunofluorescence. Several tags (FLAG, S-tag) do both.
- Do protein tags need to be removed?
- Not always — small tags like His6 are usually left on. When the tag interferes with activity, crystallization or immunogenicity, engineer a protease site (TEV, thrombin, PreScission/HRV-3C, enterokinase, or SUMO protease) between the tag and target so it can be cleaved off after purification.
- How big is a His tag?
- A 6×His tag is six histidine residues, about 0.84 kDa. His8 (eight histidines, ~1.1 kDa) binds IMAC resin more tightly and is useful for low-expression or membrane proteins.
- Why are GST, MBP and SUMO shown without a full sequence?
- These are whole proteins (hundreds of residues) fused as solubility/affinity partners, so the table lists "see notes" plus an approximate mass and the UniProt/vector source instead of inlining the full sequence. Retrieve the exact sequence from the cited UniProt accession or vector map.
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Related tools and references
Use these related pages when this table raises a practical calculation or workflow question.
Tools
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Compute molecular weight, isoelectric point, extinction coefficient and composition.
Protease Digestion tool
Digest a protein with trypsin, Lys-C, chymotrypsin and more, and get peptide masses.
Reverse Translate tool
Back-translate a protein to DNA using most-frequent or degenerate IUPAC codons.