Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Nat Protoc. Author manuscript; available in PMC 2014 March 4.
Published in final edited form as:
PMCID: PMC3941705

Site-specific N-terminal labeling of proteins using sortase-mediated reactions


For many proteins, the N- or the C-terminus make essential contributions to substrate binding, for protein-protein interactions, or for anchoring the proteins to a membrane. In other circumstances, at least one of the termini is buried within the protein, rendering it inaccessible to labeling. The possibility of selective modification of one of the protein’s termini may present unique opportunities for biochemical and biological applications. We describe sortase-mediated reactions to selectively label the N-terminus of a protein with a variety of functional groups. If sortase, the protein of interest, and a suitably functionalized label are available, the reactions usually require less than 3 hours.


Modification of proteins with fluorophores or other compounds of interest enables creation of novel biological tools to study cellular pathways and molecular mechanisms 1,2. Conjugation of a toxic moiety or antigen to a targeting antibody expands the use of these proteins for cellular delivery purposes, while reducing toxic side effects 35. The key to tagging the protein of interest without disrupting its structure or function is selective site-specific labeling. Specific labeling at the N-terminus of a protein is often the only option available, either because of the constraints imposed by the protein’s topology 68, or because the native C-terminus is essential for function (e.g.,ubiquitin) 9,10 and/or cellular membrane anchoring 11,12 ,which renders cytosolic portions of proteins inaccessible to added sortase if labeling is to be conducted on intact cells.

Maleimide and NHS-ester derived probes are commonly used to modify proteins, as they are reactive with thiol and amino-groups of cysteine and lysine, respectively 1315. While cysteines and lysines can be introduced at the N-terminus of a protein, the use of side chain-reactive probes lacks selectivity and may also compromise the active site of the protein being labeled. Genetic engineering approaches allow site-specific modification, but may interfere with protein structure 16,17. While the sortase-mediated labeling method described here overcomes many of these challenges, the possibility of unintended alterations that interfere with protein function should always be considered in design and interpretation.

Sortases are expressed by Gram-positive bacteria. They are essential in cell wall biosynthesis 1821 and covalent attachment of proteins to the peptidoglycan cell wall. Additional background on sortase enzymes and a detailed protocol for C-terminal labeling using sortases can be found in (this issue of) Nature Protocols [ref]. In the specific instance of N-terminal labeling described here, the protein to be labeled is engineered with an exposed stretch of glycines or alanines at its N-terminus when using sortase A from Staphylococcus aureus or Streptococcus pyogenes, respectively. A peptide decorated with a functional group of choice (fluorophores, biotin, lipids, nucleic acids, carbohydrates, etc) and comprising a sortaserecognition motif LPXTG/A sequence (X being any amino acid 2229) at its C-terminus is then added to the reaction together with sortase. Sortase A cleaves between the Thr and Gly/Ala residues, forming a thioester intermediate with the peptide probe. Nucleophilic attack by the Nterminally modified protein of interest resolves the intermediate, resulting in the formation of a covalent bond between the peptide probe and the N-terminus of the protein (Fig.1). Alternatively, depsi-peptides can be used for N-terminal labeling 29,30. Depsi-peptides feature an ester linkage between the threonine and glycine, instead of an amide peptide bond to yield a more effective leaving group. By using depsi-peptides, the probe concentration in the reaction can be lowered while maintaining yields.

Figure 1
N-terminal labeling of proteins


Proteins to be labeled at the N-terminus must display glycine (SrtA, S. aureus) or alanine (SrtA, S. pyogenes) residues at their N-terminus 8,23,29,31. Using standard molecular cloning methods, a stretch of one to five glycines or two to five alanines is introduced, immediately following the initial methionine, which is often removed by methionylaminopeptidase 32. The requisite number of glycines/alanines should be determined empirically, as it depends on the exposure of the N-terminus, although usually three residues suffice. A linker may be interposed between the N-terminal glycines/alanines and the remainder of the protein to improve accessibility 8.

In those cases where the initial methionine is not (completely) removed after protein synthesis, we use an alternative strategy to expose glycine residues at the N-terminus: a thrombin cleavage site (Leu-Val-Pro-Arg-Gly) is inserted to precede the glycine stretch 31,33. Thrombin cleaves between the Arg and Gly residues, thus ensuring that upon cleavage these glycines are exposed on the protein molecule to be labeled. Critical Step: Although thrombin is somewhat specific, we recommend that before choosing this option, the user confirm that the protein of interest is not itself directly susceptible to thrombin cleavage.


Many core facilities devoted to peptide synthesis can deliver modified peptides for use in N-terminal sortase-catalyzed labeling. Alternatively, commercial providers are a readily accessible source of these materials. However, manual synthesis is cost effective, expands the range of modifications possible for the individual user, and for those reasons it is included in this protocol. The following section provides a protocol for the synthesis of 5(6)-TAMRA, biotin labeled, and NHS ester linked probes for sortase A-mediated reactions. Attachment of fluorophores allows microscopy of internalized labeled proteins, while biotin attachment is especially useful for tagging proteins for pull-down experiments with streptavidin beads. For N-terminal reactions, the probe is linked to the N-terminus of a LPETGG peptide for S. aureus and a LPETAA peptide for S. pyogenes sortase A. Although any amino acid can be placed between the proline and threonine, we prefer glutamic acid or other polar amino acids to aid in precipitation of crude peptide after cleavage from the solid phase resin. To reduce the time required for synthesis and purification, Fmoc-Lys(biotin)-OH, Fmoc-Lys(5-TAMRA)-OH, and other pre-conjugated building blocks can be obtained commercially. These building blocks should be coupled to the leucine residue of the sortase recognition sequence.



  • N,N-Dimethylformamide (DMF; Applied Biosystems, cat. no. GEN002007) ! Caution Flammable/toxic
  • Acetonitrile (ACN; JT Baker Analytical, cat. no. 9017-03) ! Caution Flammable/toxic
  • N-methyl-2-pyrrolidone (NMP; Sigma Aldrich, cat. no. 328634-2L) ! Caution Flammable/irritant/toxic
  • Diisopropylethylamine (DIPEA; Fisher BioReagent, cat. no. BP592500) ! Caution Highly flammable/corrosive
  • Dichloromethane (DCM; VWR, cat. no. JT9305-3) ! Caution Carcinogen
  • Dimethylsulfoxide (DMSO; EMD Chemicals Inc, cat. no. MX1458-6) ! Caution Irritant/flammable
  • Piperidine (Sigma Aldrich, cat. no. 104094) ! Caution Flammable/corrosive
  • Pyridine (Sigma Aldrich, cat. no. 270407) ! Caution Highly flammable/toxic
  • Diethyl ether (EMD Chemicals Inc, cat. no. EX0185-8)! Caution Highly flammable/harmful
  • Trifluoroacetic acid (TFA; Sigma Aldrich, cat. no. T6508) ! Caution Strongly corrosive/toxic
  • Triisopropylsilane (TIS; Sigma Aldrich, cat. no. 233781) ! Caution Flammable.
  • 5(6)-carboxy-tetramethylrhodamine (5(6)-TAMRA; Novabiochem, cat. no, 815030)
  • Biotin (Sigma Aldrich, cat. no. B4501)
  • Fmoc-Ala-OH (Novabiochem, cat. no. 852003)
  • Fmoc-Lysine(Mtt)-OH (EMD biosciences, cat. no. 04-12-1137)
  • Fmoc-Cys(Trt)-OH (Novabiochem, cat. no. 852008)
  • Fmoc-Gly-OH (Novabiochem, cat. no. 852001)
  • Fmoc-Thr(tBu)-OH (Novabiochem, cat. no. 852000)
  • Fmoc-Glu(OtBu)-OH(Novabiochem, cat. no. 852009)
  • Fmoc-Pro-OH (Novabiochem, cat. no. 852017)
  • Fmo-Leu-OH (Novabiochem, cat. no. 852011)
  • Fmoc-ε-caproic acid (Novabiochem, cat. no. 852053)
  • 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU; Novabiochem, cat. no. 851006) ! Caution Irritant/harmful
  • Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP; Novabiochem, cat. no. 851009) ! Caution Irritant/harmful
  • Ninhydrin (Eastman, cat. no. 2495) ! Caution Harmful
  • Potassium cyanide (Sigma-Aldrich, cat. no. 31252) ! Caution Highly toxic/hazardous to the environment
  • Phenol (J.T. Baker, cat. no.2858-04) ! Caution Toxic/corrosive
  • Rink amide resin SS, 100–200 mesh, 1% DVB (Advanced Chemtech, cat. no. SA5030)


  • 3 mL syringe equipped with fritted glass filter (New England Peptide, AC0-003)
  • Glass column with a frited glass filter bottom
  • Wrist Action shaker (St. John Associate Inc.)
  • Swinging bucket centrifuge (Beckman)
  • HPLC system (Agilent 1100 series)
  • Reverse phase C18 column (Waters Delta Pak 15 µm, 100 Å, 7.8×300 mm)
  • Liquid chromatography/mass spectrometry LC/MS
  • Nuclear Magnetic Resonance (NMR) spectrometer
  • Vacuum line
  • Lyophilizer
  • Microfuge tubes
  • Test tube racks
  • Syringes
  • Graduated cylinders
  • Heating block
  • Erlenmeyer flasks
  • 50 mL polypropylene conical tubes (Corning)


  • Kaiser test solution A dissolve 500 mg of ninhydrin in 10 mL ethanol.
  • Kaiser test solution B dissolve 80 g of phenol in 20 mL of ethanol.
  • Kaiser test solution C dissolve 1.3 mg potassium cyanide (20 µmol) in 20 mL of water. Add 2 mL of the potassium cyanide solution to 100 mL of pyridine.
  • 20% piperidine in NMP mix 20 mL piperidine and 80 mL NMP.
  • Cleavage cocktail (95% TFA, 2.5% H2O, and 2.5% TIS) mix 4.75 mL TFA, 125 µL H2O and 125 µL TIS
  • Buffer A HPLC 0.1% TFA in H2O
  • Buffer B HPLC 0.1% TFA in ACN
  • Buffer A LC/MS 0.1% formic acid in H2O
  • Buffer B LC/MS 0.1% formic acid in ACN

Kaiser Test TIMING 5 min

Monitor peptide couplings by performing a Kaiser test 34.

  • 1
    Mix 2 µL of solution A, 2 µL of solution B and 4 µL of solution C in a microcentrifuge tube.
  • 2
    Add 5–10 dried beads of the rink amide resin to the mixture at room temperature.
  • 3
    Heat the tube to 95°C for 3 min. A dark blue color indicates incomplete coupling.

Note: This test works on primary amines and does not work for testing the attachment of an amino acid to a Pro residue. Alternative methods such as the acetaldehyde/p-chloroanil test or microcleavage can be used to monitor these reactions 35,36.

Microcleavage Test TIMING 45 min

  • 1
    Prepare a 30 µL solution of 95% TFA and 5% H2O.
  • 2
    Add 5–10 beads to the solution and cleave for 20 min.
  • 3
    Take 5 µL of the supernatant and dilute in 30 µL of H2O and analyze by mass spectrometry.


Note: Use Fmoc-Ala-OH in place of Fmoc-Gly-OH to make probes for S. pyogenes sortase A

Resin Preparation TIMING 15 min

  • 1
    Add 100 µmol of Rink amide resin (167 mg) into a capped glass column with a fritted glass filter bottom, solvate the resin in dichloromethane (DCM) (7 mL) by shaking for 15 min in a wrist-action shaker and remove the DCM by vacuum filtration.

Deprotection TIMING 30 min

  • 2
    Add 20% piperidine solution in N-methyl-2-pyrrolidone (NMP) (7 mL) and shake for 15 min to remove the resin’s Fmoc protecting groups.
  • 3
    Remove the piperidine solution by vacuum filtration and wash the resin three times with NMP (7 mL), three times with DCM (7 mL) and an additional time with NMP.

Coupling Reaction TIMING 2–3 h until pause point, 3.5 h per coupling cycle

  • 4
    Dissolve Fmoc-Gly-OH (89 mg, 300 µmol), HBTU (114 mg, 300 µmol), and DIPEA (104 µL, 600 µmol) in NMP (7 mL) and add to the resin. Shake the suspension for 2 h at room temperature.
  • 5
    Remove the reaction solution by vacuum filtration and wash the resin three times with NMP (7 mL) and three times with DCM (7 mL). Confirm the coupling reaction by performing a Kaiser test.
    Note: If the reaction is incomplete repeat steps 4–5 with half the amount of reagents used for a standard coupling and shake for 1 h.

PAUSE POINT: The resin can be stored at 4 °C after drying under vacuum. CRITICAL STEP: At this stage, store peptides in their Fmoc-protected form.

  • 6
    Repeat steps 1–5 with Fmoc-Gly-OH (89 mg, 300 µmol), Fmoc-Thr(OtBu)-OH (119 mg, 300 µmol), Fmoc-Glu(OtBu)-OH (127 mg, 300 µmol), Fmoc-Pro-OH (101 mg, 300 µmol), Fmoc-Leu-OH (106 mg, 300 µmol), Fmoc-ε-aminocaproic acid (85 mg, 300 µmol).
    Note: The Kaiser test does not work for verifying the extent of the Leu coupling, since the Nterminus of Pro is a secondary amine. To test this coupling reaction, one can use the chloroanil test or microcleavage. Note that the orthogonal protecting groups may not be fully removed during this abbreviated cleavage.
  • 7
    After removing the Fmoc on the ε-aminocaproic acid residue, add a solution of 5(6)-TAMRA (52 mg, 120 µmol), PyBOP (63 mg, 120 µmol), and DIPEA (42 µL, 240 µmol) and shake overnight at room temperature. To prevent quenching of the fluorophore, wrap the column in aluminum foil.
  • 8
    Repeat step 5 and perform the Kaiser test to check the TAMRA coupling.

Cleavage from Resin TIMING 3 h

  • 9
    Suspend the resin in cleavage solution consisting of 95% TFA, 2.5% H2O, and 2.5% TIS (5 mL) for 2 h at room temperature.
  • 10
    Elute the cleavage solution into 90 mL of ice cold (−20 °C) diethyl ether and rinse the resin with an additional 3 mL of the cleavage solution into the ether.
  • 11
    Store the ether solution at −20 °C for 20 min to precipitate the peptide. Centrifuge the suspension at 1,900g for 15 min at 4 °C, decant the supernatant and gently evaporate the remaining ether under reduced pressure.

Pause point: The crude peptide can be stored as a solid at −20 °C.

Critical step: Verify the identity and purity by LC/MS analysis (linear gradient 5→45% LC/MS buffer B over 10 min). If LC/MS shows that the crude peptide is of sufficient purity, the next steps (12–14) may be omitted and the peptide may be used directly in sortase reactions.

HPLC purification

  • 12
    Dissolve the dried peptide in H2O (2 mL) and centrifuge at 14,000 rpm for 10 min in a tabletop centrifuge to remove particulate matter.
    Note: Up to 50% of tert-butanol may be added to peptides that do not dissolve in pure H2O. Also spin filters or syringe filters may be used to remove particulate matter.
  • 13
    Purify the centrifuged supernatant by reverse-phase HPLC on a C18 column using a 10–75% buffer B gradient.
  • 14
    Analyze the fractions for product by LC/MS and lyophilize the desired fractions to dryness.
    Note: TAMRA containing probes consist of a mixture of regio-isomers that will likely result in two product peaks during reverse phase HPLC purification. The different isomers have no effect on labeling.

Critical step: Verify the identity and purity by LC/MS analysis (linear gradient 5→45% LC/MS buffer B over 10 min) and NMR spectroscopy.

Pause point: The lyophilized peptide can be stored at −20 °C indefinitely.

B) Biotin-LPETGG Probe

  • 1
    Use the same reaction conditions as for synthesis of the TAMRA-LPETGG probe through the Fmoc deprotection step of the Leu residue. At this point, add a solution of biotin (74 mg, 300 µmol), HBTU (114 mg, 300 µmol) and DIPEA (104 µL, 600 µmol) in NMP (7 mL); shake for 2 h.
  • 2
    Remove the reaction solution by vacuum filtration, wash the resin, and check the success of biotin coupling with a Kaiser test (remaining free amines).
  • 3
    Cleave the product from the resin as indicated in steps 9–11 for the TAMRA probe.
  • 4
    Purify by reverse phase HPLC as indicated in steps 12–14 of the TAMRA probe

Critical step: Verify the identity and purity by LC/MS analysis (linear gradient 5→45% B in 10 min) and NMR spectroscopy.

Pause point: The lyophilized peptide can be stored at −20 °C indefinitely.

C) Other LPETGG Probes

For the addition of other functional groups or acid-labile substituents, we recommend using probes conjugated via NHS ester couplings.

  • 1
    Synthesize LPETGG as described above.
  • 2
    Remove the Fmoc on the Leu according step 2 of the general method.
  • 3
    Cleave the peptide from the resin as described in steps 9–11.
  • 4
    Purify the peptide by HPLC, steps 12–14 or if of sufficient purity as indicated by LC/MS, use the crude peptide directly after precipitation from the cleavage solution into diethyl ether.
  • 5
    Dissolve the lyophilized peptide in DMSO.
  • 6
    Add three equivalents of the LPETGG peptide in DMSO to the NHS ester probe and add 5 equivalents of DIPEA.
  • 7
    Incubate the reaction for 12 h at RT.
  • 8
    Dilute the reaction to 25% DMSO with H2O and purify by HPLC as indicated above.
    Note: For fluorescent probes, protect the reaction mixture from light by covering it in aluminum foil.



The protocol for expression and purification of the various sortase A is found (elsewhere) in (this issue of) Nature Protocols.


  • Purified target oligoglycine/alanine protein in buffer (no phosphate-based buffer if a Ca2+-dependent sortase A is used)
  • Purified sortase A in buffer (no phosphate-based buffer if a Ca2+-dependent sortase A is used)
  • LPETGG- or LPETAA-based peptide probes (if using S.aureus or S.pyogenes sortase A, respectively) stock solution: 5 mM in DMSO or water (10× stock). If a polypeptide is used, then dissolve it in buffer (no phosphate-based buffer if a Ca2+-dependent sortase A is used)
  • 4x loading LDS-buffer (Invitrogen, cat. no. LC5800)
  • Common reagents for SDS-PAGE analysis (10 or 12% acrylamide gels)
  • Tris Hydrochloride, Tris-HCl (American Bioanalytical, cat. no. AB02005-05000)
  • Sodium Chloride, NaCl (American Bioanalytical, cat. no. AB01915-10000)
  • Calcium Chloride Dyhydrate, CaCl2•H2O (Mallinckrodt Chemical, cat. no. 4160)
  • Brilliant blue R (Sigma-Aldrich, cat. no. B7920)
  • Methanol, MeOH (EMD, cat. no. MX0488-1)
  • Acetic acid, AcOH (VWR, cat. no. BDH3094)
  • Ethanol, EtOH (Pharmco-AAPER, cat. no. 111000190)


  • Micropipettes
  • 1.5 mL centrifuge tubes
  • Centrifuge for 1.5 mL centrifuge tubes
  • 37 °C water bath or thermocycler
  • Equipment for SDS-PAGE, western-blot, Coomassie staining
  • Fast protein liquid chromatography (FPLC) system with size exclusion and ion exchange columns
  • Liquid chromatography/mass spectrometry (LC/MS)
  • Amicon ultra spin concentrators 10 kDa NMWL (Millipore)


  • Sortase buffer: 500 mM Tris-HCl pH 7.5, 1.5 M NaCl, 100 mM CaCl2 (not required if using a Ca2+-independent sortase) (10× stock).
  • Size exclusion chromatography buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl.
  • Coomassie blue staining: Dissolve 1.25 g Brilliant Blue R in a mixture of methanol (200 mL), water (250 mL) and acetic acid (50 mL). Store in a dark container at RT.
  • Destaining solution: Mix water, ethanol and acetic acid in a ratio of 6:3:1. Store at RT.


A) Setting up the reaction conditions TIMING 8 h

  • (i) Mix 0.5–1 mM LPETG/A-containing probe, 10–50 µM target protein, 20–150 vM sortase A in 1× sortase buffer (final concentrations). The controls to be included are: target protein only, sortase only, oligoglycine probe only, target protein and sortase, target protein and oligoglycine probe, sortase and oligoglycine probe.
  • (ii) Incubate the reactions at RT or at 37 °C. Take 1 µl aliquots at 15’, 1, 3, 5 h. Add 1× SDSgel loading buffer to the aliquots to stop the reaction and boil 2 min.

    PAUSE POINT: The 1 µl aliquots can be frozen at −20 °C until further analysis.

  • (iii) Analyze the 1 µl aliquots by SDS-PAGE (10 % or 12% acrylamide for substrates in the 12–80 kDa range) followed by Coomassie staining.

    Note: Some products and starting material proteins have similar molecular weights. A gradient gel of appropriate size and porosity may achieve better separation.

Anticipated result: A successful sortase reaction results in the formation of the acyl-enzyme intermediate. Because the concentration of the protein to be labeled is never sufficiently high to resolve all covalent sortase-peptide intermediates, the sortase-peptide intermediate will be detected in all the reactions. Also, the acyl-enzyme intermediate is rather resistant to reducing and denaturing conditions. Thus, one can detect sortase-peptide adducts by fluorescent scanning or western-blot if the LPETG/A peptide contains a tag (e.g., dye or biotin). A sortase reaction often yields a reaction product with a distinct mobility from that of the input substrate and the hydrolysis product. The ability to distinguish the various intermediates critically depends on the MW of the anticipated products and on the gel systems used to analyze them. In cases where the MW difference is too small to be detected by gel, LC/MS analysis can be used to monitor the reaction and provide an estimated yield.

(B) Purification

  • 1
    Load the sortase reaction into a spin concentrator with an appropriate MW cutoff and centrifuge to remove unreacted (low MW) probe.
  • 2
    As needed, purify the protein of interest by size exclusion chromatography using Superdex 75 or 200 resin, depending on its Stokes’ radius. Analyze fractions by SDS-PAGE and/or LC/MS.
    Note: If the protein is sufficiently pure at this point, it can be concentrated and is ready for use.
  • 3
    If further purification is needed, concentrate the product-containing FPLC fractions with a spin concentrator. Then purify the product by ion-exchange chromatography (Mono Q or Mono S resin depending on the charge distribution of the molecule of interest) with a 0 – 1 M gradient of NaCl. Analyze fractions by SDS-PAGE and/or LC/MS and concentrate the product-containing fractions for use or storage.
    Note: In those rare instances where sortase has properties similar to the product of interest, an initial Ni-NTA affinity chromatography step can be added since the sortase has a His6 tag and will bind to the resin. If the protein of interest has a His6 tag, this added purification will not work and a different tag should be used.

ProblemPossible causeSolution
No or low protein labelingNot enough LPXTG/LPXTA probe added to the reactionReaction conditions have to be determined ad-hoc. Increase the amount of probe to 10 mM and/or decrease the amount of protein nucleophile
pH of the reaction buffer not compatible with sortase activityEnsure that the pH of the reaction buffer is appropriate. Check the pH of the stock solutions. Especially the pH of the probe solutions can be low, due to residual traces of TFA. Lyophilize the probe solution multiple times with water to remove TFA. Neutralize the probe solution with aq NaHCO3 or by adding additional buffer if the pH remains too low
Proteolysis of sortaseVerify the integrity of sortase upon reaction by Coomassie staining and/or anti-His blot. The amount of sortase before and after reaction should be equal. If not, consider the presence of a contaminating protease, which most probably co-purified with the protein of interest. We recommend further purification of the protein nucleophile.
N-terminal is not adequately exposedIncrease the length of the poly glycine/alanine nucleophile. If the initial Met is not removed during protein expression a thrombin cleavage site should be added prior to the nucleophilic Gly/Ala residue.
Wrong strain of sortase is usedEnsure that you use sortase A from S. pyogenes for alanine-based nucleophiles and sortase from S. aureus for glycine modified proteins.
Sortase is inactiveTest the preparation of sortase using N-terminal modified GFP as the nucleophile. Too high concentration of DMSO in reaction mixture, originating from peptide stock solution.
Not enough sortase added to the reactionSortase concentration has to be titrated for each substrate to be labeled.
Increase the substrate concentration and/or decrease the amount of protein to be labeled
Detection of a white fluffy precipitate during the sortase-labeling reactionCa2+ precipitateDo not use phosphate buffers if working with a Ca2+-dependent sortase.
Protein precipitates during the labeling reactionHigh concentration of protein, especially when attempting protein-protein fusionsOptimize reaction temperature and incubation time. Less protein may be sufficient to achieve the same reaction yield without precipitation.
The protein of interest precipitates at high temperaturesPerform the labeling reaction at RT and extend the reaction time. 10% glycerol may also prevent precipitation
Product and starting material comigrate on gelMW of product and starting material are similarAnalyze by LC/MS or use a gradient gel


1. Guimaraes CP, et al. Identification of host cell factors required for intoxication through use of modified cholera toxin. J Cell Biol. 2011;195:751–764. [PMC free article] [PubMed]
2. Gautier A, et al. An engineered protein tag for multiprotein labeling in living cells. Chem Biol. 2008;15:128–136. [PubMed]
3. Dmochewitz L, et al. A recombinant fusion toxin based on enzymatic inactive c3bot1 selectively targets macrophages. Plos One. 2013;8:e54517. [PMC free article] [PubMed]
4. Jang JI, et al. Expression and delivery of tetanus toxin fragment C fused to the Nterminal domain of SipB enhances specific immune responses in mice. Microbiol Immunol. 2012;56:595–604. [PubMed]
5. Gellerman G, Baskin S, Galia L, Gilad Y, Firer MA. Drug resistance to chlorambucil in murine B-cell leukemic cells is overcome by its conjugation to a targeting peptide. Anticancer Drugs. 2013;24:112–119. [PubMed]
6. Baratova LA, et al. The topography of the surface of potato virus X: tritium planigraphy and immunological analysis. J Gen Virol. 1992;73(Pt 2):229–235. [PubMed]
7. Schwede A, Jones N, Engstler M, Carrington M. The VSG C-terminal domain is inaccessible to antibodies on live trypanosomes. Mol Biochem Parasit. 2011;175:201–204. [PMC free article] [PubMed]
8. Hess GT, et al. M13 bacteriophage display framework that allows sortase-mediated modification of surface-accessible phage proteins. Bioconjugate Chemistry. 2012;23:1478–1487. [PMC free article] [PubMed]
9. Reyes-Turcu FE, et al. The ubiquitin binding domain ZnF UBP recognizes the Cterminal diglycine motif of unanchored ubiquitin. Cell. 2006;124:1197–1208. [PubMed]
10. Love KR, Pandya RK, Spooner E, Ploegh HL. Ubiquitin C-Terminal Electrophiles Are Activity-Based Probes for Identification and Mechanistic Study of Ubiquitin Conjugating Machinery. Acs Chem Biol. 2009;4:275–287. [PMC free article] [PubMed]
11. Greenhalf W, Stephan C, Chaudhuri B. Role of mitochondria and C-terminal membrane anchor of Bcl-2 in Bax induced growth arrest and mortality in Saccharomyces cerevisiae. Febs Letters. 1996;380:169–175. [PubMed]
12. Kataoka T, et al. Bcl-rambo, a novel Bcl-2 homologue that induces apoptosis via its unique C-terminal extension. Journal of Biological Chemistry. 2001;276:19548–19554. [PubMed]
13. Miyadera T, Kosower EM. Receptor site labeling through functional groups. 2. Reactivity of maleimide groups. Journal of Medicinal Chemistry. 1972;15:534–537. [PubMed]
14. Palmer M, Buchkremer M, Valeva A, Bhakdi S. Cysteine-specific radioiodination of proteins with fluorescein maleimide. Anal Biochem. 1997;253:175–179. [PubMed]
15. Hnatowich DJ, et al. Labeling peptides with technetium-99m using a bifunctional chelator of a N-hydroxysuccinimide ester of mercaptoacetyltriglycine. J Nucl Med. 1998;39:56–64. [PubMed]
16. Rabuka D, Rush JS, deHart GW, Wu P, Bertozzi CR. Site-specific chemical protein conjugation using genetically encoded aldehyde tags. Nature Protocols. 2012;7:1052–1067. [PMC free article] [PubMed]
17. Wu P, et al. Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag. Proceedings of the National Academy of Sciences of the United States of America. 2009;106:3000–3005. [PubMed]
18. Paterson GK, Mitchell TJ. The biology of Gram-positive sortase enzymes. Trends Microbiol. 2004;12:89–95. [PubMed]
19. Ton-That H, Liu G, Mazmanian SK, Faull KF, Schneewind O. Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif. Proceedings of the National Academy of Sciences of the United States of America. 1999;96:12424–12429. [PubMed]
20. Mazmanian SK, Liu G, Ton-That H, Schneewind O. Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall. Science. 1999;285:760–763. [PubMed]
21. Spirig T, Weiner EM, Clubb RT. Sortase enzymes in Gram-positive bacteria. Mol Microbiol. 2011;82:1044–1059. [PMC free article] [PubMed]
22. Popp MW, Antos JM, Ploegh HL. Site-specific protein labeling via sortasemediated transpeptidation. Curr Protoc Protein Sci. 2009 Chapter 15, Unit 15 13. [PubMed]
23. Popp MW, Ploegh HL. Making and breaking peptide bonds: protein engineering using sortase. Angew Chem Int Ed Engl. 2011;50:5024–5032. [PubMed]
24. Ton-That H, Schneewind O. Anchor structure of staphylococcal surface proteins. IV. Inhibitors of the cell wall sorting reaction. J Biol Chem. 1999;274:24316–24320. [PubMed]
25. Race PR, et al. Crystal structure of Streptococcus pyogenes sortase A: implications for sortase mechanism. J Biol Chem. 2009;284:6924–6933. [PMC free article] [PubMed]
26. Tanaka T, Yamamoto T, Tsukiji S, Nagamune T. Site-specific protein modification on living cells catalyzed by Sortase. Chembiochem : a European journal of chemical biology. 2008;9:802–807. [PubMed]
27. Yamamoto T, Nagamune T. Expansion of the sortase-mediated labeling method for site-specific N-terminal labeling of cell surface proteins on living cells. Chemical Communications. 2009:1022–1024. [PubMed]
28. Tsukiji S, Nagamune T. Sortase-mediated ligation: a gift from Gram-positive bacteria to protein engineering. Chembiochem : a European journal of chemical biology. 2009;10:787–798. [PubMed]
29. Williamson DJ, Fascione MA, Webb ME, Turnbull WB. Efficient N-terminal labeling of proteins by use of sortase. Angew Chem Int Ed Engl. 2012;51:9377–9380. [PubMed]
30. Cudic P, Stawikowski M. Peptidomimetics: Fmoc solid-phase pseudopeptide synthesis. Methods Mol Biol. 2008;494:223–246. [PubMed]
31. Antos JM, et al. Site-specific N- and C-terminal labeling of a single polypeptide using sortases of different specificity. Journal of the American Chemical Society. 2009;131:10800–10801. [PMC free article] [PubMed]
32. Barrett AJ, Rawlings ND, Woessner JF. Handbook of proteolytic enzymes. Academic Press; 1998.
33. Liu D, Xu R, Dutta K, Cowburn D. N-terminal cysteinyl proteins can be prepared using thrombin cleavage. Febs Letters. 2008;582:1163–1167. [PMC free article] [PubMed]
34. Kaiser E, Colescott RL, Bossinger CD, Cook PI. Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal Biochem. 1970;34:595–598. [PubMed]
35. Vojkovsky T. Detection of secondary amines on solid phase. Pept Res. 1995;8:236–237. [PubMed]
36. Gisin BF. The monitoring of reactions in solid-phase peptide synthesis with picric acid. Anal Chim Acta. 1972;58:248–249. [PubMed]