Tags compared

Protein tags for surface immobilisation, compared

Most ways of putting a protein on a surface fall into a few architectures: let it adsorb, couple it chemically, give it a tag that a surface-bound partner captures, fuse it to a domain that reacts with a surface ligand, or ligate it enzymatically. Each solves orientation and retention to a different degree, and each asks something of the surface, the protein or the workflow. This page describes the common engineered tag systems at that level, and states what changes with PepLock. Chemical coupling and adsorption are covered on the Technology page.

PepLock appears two ways in what follows. A PepLock-tagged protein, binder or peptide attaches directly to the assay surface. A PepLock-tagged binder attaches to a purification resin or membrane and serves as the capture agent there. PepLock attachment is generally not reversible; where a protein needs a defined release point, a cleavage tag between the anchor and the protein provides it.

At a glance

What each system adds, and where

Engineered tag systems compared with PepLock
System	Added to the protein	Needed on the surface	Added to the reaction	Attachment
His-tag / Ni-NTA	Short polyhistidine tag	Chelator and nickel or cobalt	None	Reversible by design
Strep-tag II and Twin-Strep-tag	Short peptide tag	Strep-Tactin protein	None	Reversible with biotin or desthiobiotin
Epitope tags: FLAG, HA, myc, V5, ALFA-tag and Spot-tag	Short peptide tag	Antibody or nanobody	None	Reversible by elution
Protein A, Protein G and anti-Fc capture	An Fc, if the binder lacks one	Protein A, Protein G or anti-Fc antibody	None	Reversible at low pH
Biotin / streptavidin, including AviTag	Biotin, chemically or via AviTag	Streptavidin or avidin	Biotin ligase, ATP and biotin for AviTag	Very tight, non-covalent
SpyTag/SpyCatcher, with SnoopTag and DogTag	Short peptide tag	Catcher protein	None	Spontaneous covalent
HaloTag	Large fusion domain	Chloroalkane ligand	None	Covalent
SNAP-tag and CLIP-tag	Large fusion domain	Benzylguanine or benzylcytosine ligand	None	Covalent
GST and MBP	Large fusion domain	Glutathione or amylose	None	Reversible by elution
Sortase	Short recognition motif	Oligoglycine	Sortase enzyme, calcium	Covalent, reversible under reaction conditions
Material-binding peptides	Short peptide	The bare material itself	None	Non-covalent, material-specific
PepLock	Short peptide anchor	The intended assay surface	None	Withstands washes, detergents, salts, acids, bases and chaotropes

Scroll the table sideways to see every column.

Family 1

Capture tags: a surface-bound protein holds the tag

His-tag / Ni-NTA

What it is: A polyhistidine tag on the protein and a nickel-chelate surface.
What it needs: Capture is designed to release under imidazole, EDTA and other chelators, reducing agents or low pH, which suits purification but not a surface that must hold a protein through repeated use or sample matrices containing those agents. Protein and metal can leach from the surface over time, and histidine-rich proteins can bind non-specifically.
Where it is used: Purification first; also Ni-NTA plates and biosensor chips for assay loading and interaction measurements where the protein will be released or the surface regenerated.
Main drawback: Capture is designed to let go, so imidazole, chelators, reducing agents or low pH in a sample or wash release the protein, and metal can leach from the surface.
With PepLock: Attachment withstands washes, detergents, salts, acids, bases and chaotropes; PepLock is compatible with a His6 tag kept for purification.

Strep-tag II and Twin-Strep-tag

What it is: Strep-tag II is a short peptide tag, and Twin-Strep-tag is two copies linked in one sequence, both captured by Strep-Tactin, an engineered streptavidin, coated on the surface.
What it needs: The Strep-Tactin layer is a protein that has to be produced and itself immobilised on every surface, usually by chemical coupling, before the tagged protein is added. Capture is designed to be reversible: biotin or desthiobiotin displaces the tag, which suits purification and regenerable biosensor chips.
Where it is used: Protein purification, and capture on Strep-Tactin-coated plates and chips for binding and immunoassay formats.
Main drawback: Every surface needs Strep-Tactin produced and coupled on first, and free biotin in a sample competes for the same site and can release the tag.
With PepLock: A Strep-tagged protein that also carries a PepLock anchor is purified on Strep-Tactin as usual, then attaches directly to the assay surface.

Epitope tags: FLAG, HA, myc, V5, ALFA-tag and Spot-tag

What it is: An epitope tag is a short peptide, typically eight to fourteen amino acids, recognised by a specific antibody; ALFA-tag and Spot-tag are recognised by nanobodies instead.
What it needs: For immobilisation, the antibody or nanobody is coated on the surface first and captures the tagged protein, so every surface carries an antibody layer that itself has to be attached and oriented. Capture is reversible by competing peptide or low pH, which suits pull-downs and elution but means retention depends on the antibody's hold under the assay conditions.
Where it is used: Detection and pull-downs above all; anti-FLAG and anti-HA plates, beads and resins are sold for capture and purification.
Main drawback: Retention depends on an antibody's hold, and that antibody layer has to be immobilised and oriented on every surface before the tagged protein is captured.
With PepLock: A PepLock-tagged protein keeps its FLAG or HA tag for detection and attaches directly to the assay surface with consistent orientation.

Protein A, Protein G and anti-Fc capture

What it is: Protein A and Protein G bind the constant (Fc) region of antibodies, so an antibody or Fc-fusion is captured by its Fc when the surface is coated with Protein A, Protein G or an anti-Fc antibody.
What it needs: Every surface carries a capture protein, which has to be immobilised and qualified, and only Fc-bearing molecules can be captured. Binders that lack an Fc, such as scFvs and nanobodies, are often fused to one for capture; the Fc dimerises the binder, and the resulting bivalency can confound avidity with true affinity in binding measurements. Capture is oriented, with the binding sites facing away from the surface, and is reversible at low pH, which is how many chips and columns are regenerated.
Where it is used: Antibody immobilisation on biosensor chips, plates and beads, and antibody purification.
Main drawback: Samples that contain antibodies, such as serum or plasma, compete for the same Protein A or G sites, so the format is unsuited to many diagnostic matrices.
With PepLock: A PepLock-tagged nanobody or scFv attaches directly to the assay surface as the capture agent, as a monomer and with consistent orientation.

Family 2

Biotin and streptavidin, including AviTag

Biotin / streptavidin, including AviTag

What it is: Biotinylation of the molecule, either chemically or at a single site through an AviTag and the biotin ligase BirA, and a surface coated with streptavidin, a capture protein.
What it needs: Every surface carries a second protein, adding cost and another component to qualify. Chemical biotinylation is not site-specific; enzymatic biotinylation is, but adds an enzyme, biotin and ATP, and a step to remove free biotin.
Where it is used: Assay capture, affinity isolation and interaction measurements across diagnostics and research; one of the most established capture systems.
Main drawback: Both partners have to be installed, biotin on the molecule and streptavidin on the surface, and free biotin or biotinylated proteins in a sample can interfere.
With PepLock: No capture protein and no biotinylation step: a PepLock-tagged molecule attaches without a capture protein, site-specifically, in one aqueous step.

Family 3

Tag and Catcher pairs: a partner protein locks the tag

SpyTag/SpyCatcher, with SnoopTag and DogTag

What it is: SpyTag/SpyCatcher and other isopeptide tag pairs join a short peptide tag to a partner protein, which has to be produced, purified and placed on the surface first. The pair forms a spontaneous covalent isopeptide bond on mixing, with no enzyme added; SpyTag003/SpyCatcher003 is the current generation, with faster reaction and compatibility with earlier Spy versions.
What it needs: For immobilisation, SpyCatcher is immobilised first, usually by chemical coupling or through its own tag, and the SpyTag-fused protein is then added; the fusion has to be tolerated by the protein and tested. The partner protein adds a protein layer, its production and its cost to every surface.
Where it is used: Protein assembly, vaccine nanoparticle decoration, hydrogels and research immobilisation; the best known of the pairs.
Main drawback: SpyCatcher is a partner protein that has to be expressed, purified and immobilised on every surface before the tagged protein can be added.
Related systems: SnoopTag/SnoopCatcher is an orthogonal pair that reacts with its own partner and not with Spy, so two components can be assembled on one surface or scaffold independently. DogTag/DogCatcher is a further orthogonal pair whose tag tolerates placement inside loops of a folded protein, where terminal tags are not available. Split inteins work on the same partner-on-the-surface principle: each side carries half an intein, and on contact the halves splice out to leave a native peptide bond, with no enzyme added but a protein half to produce and place on every surface.
With PepLock: No partner protein and no catalyst: a PepLock-tagged molecule attaches in one aqueous step, with consistent orientation.

Family 4

Fusion domains: a protein domain meets a small-molecule surface ligand

HaloTag

What it is: HaloTag is an engineered bacterial dehalogenase, a domain of roughly thirty kilodaltons fused to the protein, that forms a covalent bond with a synthetic chloroalkane ligand.
What it needs: For immobilisation the chloroalkane ligand is installed chemically on beads, plates or chips, and the HaloTag fusion then attaches to it without an added enzyme. The domain is a substantial addition to the protein, and every surface needs the ligand chemistry installed and qualified.
Where it is used: Live-cell imaging above all, with established resin capture, purification and protein-interaction workflows.
Main drawback: A domain of roughly thirty kilodaltons added to every protein, and a chloroalkane ligand that has to be chemically installed on every surface.
With PepLock: A PepLock-tagged protein attaches directly to a plate, bead, membrane or electrode through a short peptide anchor built into its sequence, in one aqueous step.

SNAP-tag and CLIP-tag

What it is: SNAP-tag is an engineered alkylguanine transferase domain of roughly twenty kilodaltons that reacts covalently with benzylguanine substrates; CLIP-tag is a variant that reacts with benzylcytosine, so the two can be used together.
What it needs: Substrates can carry dyes or biotin or be fixed to a surface; immobilisation needs a support carrying the matching substrate, which is a custom preparation rather than a standard assay plate.
Where it is used: Protein labelling and imaging in vitro and in cells; surface immobilisation is possible but is not the main use.
Main drawback: Built for labelling: a surface carrying the matching substrate is a custom preparation, and the domain is a large addition to the protein.
With PepLock: A PepLock-tagged protein attaches directly to a plate, bead, membrane or electrode through a short peptide anchor built into its sequence, in one aqueous step.

GST and MBP

What it is: Glutathione S-transferase and maltose-binding protein are large fusion partners, used mainly to improve expression and for purification on glutathione or amylose surfaces; capture is reversible by elution with glutathione or maltose.
What it needs: GST-coated plates and beads support pull-downs and binding assays, with the caveats that GST dimerises and both domains add considerable bulk to the protein.
Where it is used: Expression and purification; GST-coated plates and beads for pull-downs and binding assays.
Main drawback: Large domains that add bulk, with GST also dimerising, and capture that is reversible by design, meant for purification rather than a surface that must hold.
With PepLock: A GST- or MBP-fused protein is purified as usual; with a PepLock anchor in its sequence it then attaches directly to the assay surface.

Family 5

Enzyme ligation: sortase

Sortase

What it is: Sortase A recognises a short LPXTG motif on the protein and ligates it to an oligoglycine presented on the surface, usually with calcium present, forming a native peptide bond.
What it needs: Sortase-mediated ligation joins a tagged protein to a surface carrying the partner motif, with the sortase enzyme added to the reaction and removed afterwards; the ligation is reversible, so conditions have to be managed.
Where it is used: Site-specific labelling, antibody-drug and protein-protein conjugation, and surface immobilisation in research settings.
Main drawback: The ligation is reversible under reaction conditions, so yield plateaus unless engineered around, and the enzyme has to be added and then removed.
Related systems: Microbial transglutaminase (joining a glutamine in a short tag to a surface amine) and the aldehyde tag, in which formylglycine-generating enzyme converts a motif cysteine to an aldehyde for ligation, are two further enzymatic routes, each adding an enzyme step and a matched surface chemistry.
With PepLock: No partner protein and no catalyst: a PepLock-tagged molecule attaches in one aqueous step, with consistent orientation.

Family 6

Material-binding peptides: a peptide that binds the bare material

Material-binding peptides

What it is: Material-binding peptides are short sequences, often found by display selection, that bind a particular material directly: silica, gold, polystyrene, titanium and others, and cellulose through the larger cellulose-binding domains.
What it needs: They need nothing on the surface beyond the material itself and no added reagent, which is their appeal. Binding is non-covalent and generally selective for one material; affinity varies widely between peptides and materials, and retention under washes, detergents and sample matrices has to be established case by case.
Where it is used: Biomineralisation and materials research, enzyme immobilisation on cellulose and silica, and surface functionalisation studies.
Main drawback: Binding is non-covalent and specific to one material, so retention has to be established peptide by peptide and surface by surface.
With PepLock: PepLock is a family of short peptide anchors that give proteins, peptides, antibodies and enzymes built-in immobilisation on solid surfaces. Attachment withstands washes, detergents, salts, acids, bases and chaotropes. Demonstrated on magnetic (carboxylated) beads, treated polystyrene plates and beads, nitrocellulose strips and membranes, polycarbonate and other functional plastics, gold microspheres and carbon-ink electrodes. PepLock-tagged molecules do not appreciably bind untreated polypropylene, polyethylene or polystyrene labware, so they handle like any other reagent through tubes, tips and storage plates. Attachment occurs on the intended assay surface, whether that is a treated plate, a bead, nitrocellulose or another functional polymer.

In short

What each system trades, and what changes

Every system above trades something: a protein layer on the surface, an enzyme in the reaction, a large domain on the protein, or retention that depends on conditions. With PepLock the attachment step is one aqueous step: the tagged molecule is added to the intended surface and attaches site-specifically, with consistent orientation. The Technology page sets out what the evidence covers; the data pack has the detail.

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