Protein Labeling

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The attachment of a reporter group (label) is often required for the detection and biochemical/cellular characterization of proteins and its binding partners.

Different labeling techniques & types of label such as fluorophores, biotin and luminescent dyes are available for non-radioactive protein labeling, both random and site-directed (N- or C-terminal).

Choose the appropriate labeling strategy (labeling technique & type of label) that fits your final application based on your initial protein source (Tab. 1).

Table 1: Protein Labeling Selection Matrix

Type of Label
Fluorophore Biotin Desthiobiotin CLICK functionality
Protein Source Purified protein Amine Labeling Amine Labeling Amine Labeling → Click Chemistry random Labeling Position
Thiol Labeling Thiol Labeling → Click Chemistry
Proliferating cells (Nascent proteins) Amino acid-based Metabolic Labeling
Puromycin-based Metabolic Labeling site directed
ATP dependent Lysine-based Labeling ATP dependent Lysine-based Labeling
Protein encoding DNA sequence (Cloning & cell-free expression required) dC-Puromycin-based Co-translational Labeling dC-Puromycin-based Co-translational Labeling

Labeling of Purified Proteins

Fluorescent Amine Labeling

NHS Ester of Fluorescent Dyes enable one-step labeling & detection of both proteins (e.g. antibodies) via targeting of Lysine and any other primary amine-containing macromolecules.

In general, Thiol Labeling is more specific than Amine Labeling due to the relatively low abundance of cysteines compared to lysines. Since however, appearance of cysteines and lysines varies among different proteins, the amino acid sequence and tertiary structure of the protein of interest must be analyzed prior to deciding on the labeling strategy. The intended application of the fluorescent protein also determines the labeling strategy. Amine labeling is used for immunochemistry whereas Thiol labeling is preferred for investigating structure, function and protein interaction.

NHS Ester of Alexa Fluor (AF) dyes are available as well.

Emission Colour Products Abs max. [nm] Em max. [nm] Replacement for
blue-green Atto 425 Protein Labeling Kit for primary amino groups 436 484 DEAC: 430 / 477 nm
green Atto 488 Protein Labeling Kit for primary amino groups 501 523 Alexa Fluor 488: 495 / 519 nm
Fluorescein (FITC): 495 / 520 nm
FAM: 495 / 520 nm
Oregon Green 514: 506 / 526 nm
Rhodamine green: 503 / 528 nm
Rhodamine 123: 507 / 529 nm
yellow-green Atto 532 Protein Labeling Kit for primary amino groups 532 553 Alexa Fluor 532: 531 / 554 nm
Bodipy 530/550: 534 / 554 nm
yellow- Cy3 Protein Labeling Kit for primary amino groups 550 570
yellow Atto 550 Protein Labeling Kit for primary amino groups 554 576 Alexa Fluor 555: 555 / 565 nm
Cy 3: 550 / 570 nm
Alexa Fluor 546: 556 / 573 nm
TAMRA: 546 / 576 nm
Rhodamine Red: 560 / 580 nm
Spectrum Orange: 559 / 588 nm
orange Texas Red Protein Labeling Kit for primary amino groups 583 603 Rhodamine ITC: 572 / 596 nm
Cy 3.5: 581 / 596 nm
ROX: 576 / 601 nm
Alexa Fluor 568: 578 / 603 nm
orange Atto 590 Protein Labeling Kit for primary amino groups 594 624 Alexa Fluor 594: 590 / 617 nm
Alexa Fluor 610: 612 / 628 nm
red Atto 647N Protein Labeling Kit for primary amino groups 644 669 Alexa Fluor 647: 650 / 665 nm
Cy 5: 643 / 667 nm
red Cy5 Protein Labeling Kit for primary amino groups 649 670
near-IR Atto 655 Protein Labeling Kit for primary amino groups 663 684 Alexa Fluor 660: 664 / 691 nm
Cy 5.5: 675 / 694 nm
Products & Ordering
Atto 425 Protein Labeling Kit FP-201-425 Fluorescent labeling of primary amino groups Atto 488 Protein Labeling Kit FP-201-488 Fluorescent labeling of primary amino groups
Atto 532 Protein Labeling Kit FP-201-532 Fluorescent labeling of primary amino groups Cy3 Protein Labeling Kit FP-201-CY3 Fluorescent labeling of primary amino groups
Atto 550 Protein Labeling Kit FP-201-550 Fluorescent labeling of primary amino groups Texas Red Protein Labeling Kit FP-201-TXR Fluorescent labeling of primary amino groups
Atto 590 Protein Labeling Kit FP-201-590 Fluorescent labeling of primary amino groups Atto 647N Protein Labeling Kit FP-201-647N Fluorescent labeling of primary amino groups
Cy5 Protein Labeling Kit FP-201-CY5 Fluorescent labeling of primary amino groups Atto 655 Protein Labeling Kit FP-201-655 Fluorescent labeling of primary amino groups
ROX Protein Labeling Kit FP-201-ROX Fluorescent labeling of primary amino groups

Fluorescent Thiol Labeling

Maleimides of Fluorescent Dyes enable one-step labeling & detection of both proteins (e.g. antibodies) and any other Thiol-containing macromolecules.

In general, Thiol Labeling is more specific than Amine Labeling due to the relatively low abundance of cysteines compared to lysines. Since however, appearance of cysteines and lysines varies among different proteins, the amino acid sequence and tertiary structure of the protein of interest must be analyzed prior to deciding on the labeling strategy. The intended application of the fluorescent protein also determines the labeling strategy. Amine labeling is used for immunochemistry whereas Thiol labeling is preferred for investigating structure, function and protein interaction.

Maleimides of Alexa Fluor (AF) dyes are available as well.

Emission Color Products Abs max. [nm] Em max. [nm] Replacement for
blue-green Atto 425 Protein Labeling Kit for thiol groups 436 484 DEAC: 430 / 477 nm
green Atto 488 Protein Labeling Kit for thiol groups 501 523 Alexa Fluor 488: 495 / 519 nm
Fluorescein (FITC): 495 / 520 nm
FAM: 495 / 520 nm
Oregon Green 514: 506 / 526 nm
Rhodamine green: 503 / 528 nm
Rhodamine 123: 507 / 529 nm
yellow-green Atto 532 Protein Labeling Kit for thiol groups 532 553 Alexa Fluor 532: 531 / 554 nm
Bodipy 530/550: 534 / 554 nm
yellow Atto 550 Protein Labeling Kit for thiol groups 554 576 Alexa Fluor 555: 555 / 565 nm
Cy 3: 550 / 570 nm
Alexa Fluor 546: 556 / 573 nm
TAMRA: 546 / 576 nm
Rhodamine Red: 560 / 580 nm
Spectrum Orange: 559 / 588 nm
orange Texas Red Protein Labeling Kit for thiol groups 583 603 Rhodamine ITC: 572 / 596 nm
Cy 3.5: 581 / 596 nm
ROX: 576 / 601 nm
Alexa Fluor 568: 578 / 603 nm
orange Atto 590 Protein Labeling Kit for thiol groups 594 624 Alexa Fluor 594: 590 / 617 nm
Alexa Fluor 610: 612 / 628 nm
red Atto 647N Protein Labeling Kit for thiol groups 644 669 Alexa Fluor 647: 650 / 665 nm
Cy 5: 643 / 667 nm
near-IR Atto 655 Protein Labeling Kit for thiol groups 663 684 Alexa Fluor 660: 664 / 691 nm
Cy 5.5: 675 / 694 nm
Products & Ordering
Atto 425 Protein Labeling Kit FP-202-425 Fluorescent labeling of thiol groups Atto 488 Protein Labeling Kit FP-202-488 Fluorescent labeling of thiol groups
Atto 532 Protein Labeling Kit FP-202-532 Fluorescent labeling of thiol groups Atto 550 Protein Labeling Kit FP-202-550 Fluorescent labeling of thiol groups
Texas Red Protein Labeling Kit FP-202-TXR Fluorescent labeling of thiol groups Atto 590 Protein Labeling Kit FP-202-590 Fluorescent labeling of thiol groups
Atto 647N Protein Labeling Kit FP-202-647N Fluorescent labeling of thiol groups Atto 655 Protein Labeling Kit FP-202-655 Fluorescent labeling of thiol groups

Biotin Amine Labeling

NHS Esters of Biotin enable Biotin labeling of both proteins (e.g. antibodies) via targeting of Lysine and any other primary amine-containing macromolecules. These biotinylated molecules are subsequently detected via fluoresecent or HRP-labeled streptavidin.

Binding of Biotin by avidin, streptavidin or NeutrAvidinTM is the strongest known biological interaction with a dissociation constant in the range of 10-15 M. The vitamin Biotin may be conjugated to many proteins without loss of their biological activity due to the small size of the Biotin molecule.

Products & Ordering
Biotin Protein Labeling Kit FP-320 Protein Biotinylation Kit Biotin-X Protein Labeling Kit FP-321 Protein Biotinylation Kit Biotin-XX Protein Labeling Kit FP-322 Protein Biotinylation Kit

Labeling of Nascent Proteins

ATP dependent Lysine-based Labeling

Acyl-ATP probes for identification of ATP-binding proteins in native proteomes

The comprehensive identification of ATP-binding proteins, such as the important class of kinases, and the dynamic analysis of nucleotide-protein interactions at the proteomic scale are fundamental for better understanding of the regulatory mechanisms of nucleotide-binding proteins[1].

ATP binding activities as well as specific binding sites of proteins can be detected globally in any biological sample or tissue from any species by using Biotin/Desthiobiotin-conjugated acyl-ATP probes (AcATP)[2-6].

The method is based on the following steps:

  • AcATP binds to the ATP pocket of ATP binding proteins and places the acyl group in close proximity to conserved lysine residues.
  • Nucleophilic attack by the lysine amino group, resulting in a covalent attachment of the acyl reporter and concomitant release of ATP.
  • Enrichment of Biotin/Desthiobiotin-tagged-proteins on streptavidin-coated solid support.
  • Digestion with proteases, isolation/purification of labeled peptides and identification / quantification using LC-MS/MS.
Products & Ordering
ATP-acetyl-hex-Biotin NU-277 Biotin-ATP probe Biotin-hex-acyl-ATP (BHAcATP) ATP-acetyl-Desthiobiotin NU-276 Desthiobiotin-ATP probe Desthiobiotin-acyl-ATP (DBAcATP)

Selected references:

[1] Manning et al. (2002) The protein kinase complement of the human genome. Science 298:1912.
[2] Villamor et al. (2013) Profiling protein kinases and other ATP binding proteins in Arabidopsis using Acyl-ATP probes. Molecular & Cellular Proteomics 12 (9):2481.
[3] Xiao et al. (2013) Proteome-wide discovery and characterizations of nucleotide-binding proteins with affinity-labeled chemical probes. Anal. Chem. 85 (6):3198.
[4] Okerberg et al. (2019) Chemoproteomics using nucleotide acyl phosphates reveals an ATP binding site at the dimer interface of procaspase-6. Biochemistry 58 (52):5320.
[5] Nordin et al. (2015) ATP acyl phosphate reactivity reveals native conformations of Hsp90 paralogs and inhibitor target engagement. Biochemistry 54 (19):3024.
[6] Adachi et al. (2014) Proteome-wide discovery of unknown ATP-binding proteins and kinase inhibitor target proteins using an ATP probe. J. Proteome Res. 13 (12):5461.

Amino Acid-based Fluorescent & Biotin Labeling

The dynamics of global protein synthesis (both spatial and temporal) is an essential parameter to characterize the cellular response under various physiological and pathological conditions. Protein synthesis has traditionally been monitored by metabolic labeling with 35S-methionine that competes with natural methionine for the incorporation into newly synthesized proteins. The detection is limited to autoradiography.

The cell-permeable methionine-analogs 4-Azido-L-homoalanine (L-AHA) and L-Homopropargylglycine (HPG) provide a non-radioactive alternative to analyze the global protein synthesis in cell culture. They are randomly incorporated instead of methionine during translation leading to full-length, azide-labeled or alkyne-modified proteins, respectively[1,2,3](Fig. 1).

Figure 1: Methionine analogs are suitable to incorporate azide groups (L-AHA) or alkyne groups (L-HPG) into nascent proteins. Visualization of the incorporated azide groups (L-AHA-based labeling) is performed via Cu(I)-catalyzed Click Chemistry with Alkynes of fluorescent dyes → Click Chemistry or Alkynes of Biotin → Click Chemistry or via Cu-free Click Chemistry with DBCO-containing fluorescent dyes → Click Chemistry or DBCO-containing Biotin → Click Chemistry. Visualiation of Alkyne groups (HPG-based labeling) can be performed with Cu(I)-catalayzed Click Chemistry only.

 

Search Auxiliary Cu(I) CLICK Reagents → Click Chemistry.

Selected References

[1] Dieck et al. (2012) Metabolic Labeling with Noncanonical Amino Acids and Visualisation by Chemoselective Fluorescent Tagging. Current Protocols in Cell Biology 7:7.11.1.
[2] Kiick et al. (2002) Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation. Proc. Natl. Acad. Sci. USA 99 (1):19.
[3] Dieterich et al. (2010) In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons. Nature Neuroscience 13 (7):897.

Products & Ordering
4-Azido-L-homoalanine HCl (L-AHA) CLK-AA005 (S)-2-Amino-4-azidobutanoic acid hydrochloride L-Homopropargylglycine (L-HPG) CLK-1067

Puromycin-based Fluorescent & Biotin Labeling

The dynamics of global protein synthesis (both spatial and temporal) is an essential parameter to characterize the cellular response under various physiological and pathological conditions. Current studies of the cellular protein level rely on indirect methods such as DNA and mRNA microarrays or classical radioactive metabolic labeling with 35S-methionine.

Liu et al. reported a non-radioactive alternative to analyze newly synthesized proteins in cell culture and whole organisms that is based on an alkyne analog of puromycin (Fig. 1). The cell-permeable O-propargyl-puromycin (Fig. 1A) incorporates into the C-terminus of translating polypeptide chains thereby stopping translation.

The resulting C-terminal alkyne labeled proteins can be detected via Cu(I)-catalyzed click chemistry that offers the choice to introduce a Biotin group (Azides of Biotin → Click Chemistry) for subsequent purification tasks or a fluorescent group (Azides of fluorescent dyes → Click Chemistry) for subsequent microscopic imaging[1,2,3].

In contrast to previously reported non-radioactive methionine analog-approaches[4,5], methionine free-medium is not required for O-propargyl-puromycin-based monitoring of nascent protein synthesis.

Figure 1: O-propargyl-puromycin labels newly synthesized proteins in cell culture and whole organisms (modified according to [1]. A) Chemical structure of O-propargyl-puromycin (OP-Puro). Visualization of incorporated OP-puro is performed via Cu(I)-catalyzed Click Chemistry (CuAAC) with Azides of fluorescent dyes → Click Chemistry or Azides of Biotin → Click Chemistry. B) Nascent protein expression in crypts of mouse small intestine was visualized by whole-mount staining. O-Propargyl-puromycin labeled proteins were detected with Tamra-azide (red) and nuclear DNA was stained with OliGreen (green) (modified according to [1]) C) Newly synthesized proteins in NIH3T3 cells are rapidly detected by incubation with 50 µM OP-puro. OP-puro labeled proteins have been visualized by Alexa568-azide, nuclear DNA was stained with Hoechst dye (modified according to [1]).

 

Search Auxiliary Cu(I) CLICK Reagents → Click Chemistry.

Selected References

[1] Liu et al. (2012) Imaging protein synthesis in cells and tissues with an alkyne analog of puromycin. Proc. Natl. Acad. Sci. USA 109 (2):413.
[2] Goodman et al. (2012) Imaging of protein synthesis with puromycin. Proc. Natl. Acad. Sci. USA 109 (17):E989.
[3] Salic et al. (2012) Reply to Goodman et al.: Imaging protein synthesis with puromycin and the subcellular localization of puromycin-polypeptide conjugates. Proc. Natl. Acad. Sci. USA 109 (17):E990.
[4] Dieterich et al. (2010) In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons. Nature Neuroscience 13 (7):897.
[5] Dieck et al. (2012) Metabolic Labeling with Noncanonical Amino Acids and Visualisation by Chemoselective Fluorescent Tagging. Current Protocols in Cell Biology 7:7.11.1.

Products & Ordering
O-Propargyl-puromycin NU-931

Reporter Protein-based Labeling

dC-Puromycin-based Fluorescent & Biotin Protein Labeling

Labeled-dC-Puromycin conjugates for specific C-terminal protein labeling in vitro

The antibiotic puromycin is a structural analog of aminoacyl-tRNA that has been traditionally used as an inhibitor of protein synthesis since it specifically incorporates at the C-terminus of nascent polypeptide chains thereby stopping translation. Based on this observation, a new and significantly faster protein labeling approach has been developed:

C-terminal labeled full-length protein can be produced by in vitro translation in the presence of low concentrations of labeled dC-puromycin conjugates[1].

These proteins have been successfully used to analyze protein-protein interactions (pull-down assay, FCCS, protein-protein microarrays) and protein-DNA interactions (DNA microarrays)[2,3,4].
In contrast to traditionally applied posttranslational labeling approaches, additional time consuming purification steps can be avoided due to the synchronization of protein expression and labeling.

Products & Ordering
6-FAM-dC-puromycin NU-925-6FM Biotin-dC-puromycin NU-925-BIO

Selected References

[1] Miyamoto-Sato et al. (2000) Specific bonding of puromycin to full-length protein at the C-terminus. Nucleic Acids Res. 28 (5):1176.
[2] Nemoto et al. (1999) Fluorescence labeling of the C-terminus of proteins with a puromycin analogue in cell-free translation systems. FEBS Letters 462:43.
[3] Doi et al. (2002) Novel Fluorescence Labeling and High-Throughput Assay Technologies for In Vitro Analysis of Protein Interactions. Genome Research 12:487.
[4] Kawahashi et al. (2007) High-throughput fluorescence labeling of full-length cDNA products based on a reconstituted translation system. J. Biochem. 141 (1):19.

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