We prepare a wide array of biotin and desthiobiotin conjugates, all of which are included in this section's product list. We will also custom-conjugate biotin, desthiobiotin (DSB-X), fluorophores or other haptens to proteins or other biomolecules of interest; contact Invitrogen Custom Services to request a quote.

Fluorescent Biotin Derivatives

Fluorescein Biotin

Fluorescein biotin (B1370, structure) was developed as an alternative to radioactive biotin for detecting and quantitating biotin-binding sites by either fluorescence or absorbance.ref A fluorescence polarization–based assay that employs competitive binding of fluorescein biotin to assess the degree of protein biotinylation has been reported ref (Fluorescence Polarization (FP)—Note 1.4). A similar derivative was used for determining avidin and biotin concentrations by fluorescence depolarization.ref

Biotin-4-Fluorescein

Our biotin-4-fluorescein (B10570, structure) offers a substantially improved method for quantitating biotin-binding sites. Biotin-4-fluorescein binds to avidin much faster than does conventional fluorescein biotin, allowing for rapid analysis.ref The strong quenching associated with avidin binding to biotin-4-fluorescein can be used to accurately measure the concentration of avidin or streptavidin ref (Figure 4.3.1). Engineered single-chain dimers created by circular permutation of wild-type streptavidin exhibit substantial binding preference for biotin-4-fluorescein relative to biotin.ref

Quantitation of biotin-binding sites  

 

Figure 4.3.1 Quantitation of biotin-binding sites with 8 nM biotin-4-fluorescein (B10570). Both the fluorescence and absorbance of biotin-4-fluorescein are quenched upon binding to one of the four biotin-binding sites of streptavidin (S888, pink), avidin (A887, A2667; dark blue), or the streptavidin conjugates of the Alexa Fluor 633 dye (S21375, orange) and alkaline phosphatase (S921, light blue). As a result, when a known concentration of biotin-4-fluorescein is added to a known amount of streptavidin, one can estimate the number of biotin-binding sites.

Other Fluorescent Biotin Derivatives

In addition to supplying nonfluorescent biocytin (ε-biotinoyl-L-lysine, B1592, Biotinylation and Haptenylation Reagents—Section 4.2), we offer:

  • Alexa Fluor 488 biocytin (A12924, structure)
  • Alexa Fluor 546 biocytin (A12923)
  • Alexa Fluor 594 biocytin (A12922, structure)
  • Lucifer yellow cadaverine biotin-X (L2601)
  • Lucifer yellow biocytin (L6950)
  • Oregon Green 488 biocytin (O12920)
  • Tetramethylrhodamine biocytin (T12921)

Each of these reagents contains both a fluorophore and biotin moiety in the same molecule. As with fluorescein biotin and biotin-4-fluorescein, which are described above, these reagents can be employed for detecting and quantitating biotin-binding proteins, but their principal application is as aldehyde-fixable polar cell tracers and as tracers for cell–cell communication ref (Polar Tracers—Section 14.3). Our lucifer yellow cadaverine biotin-X is reportedly well retained in aldehyde-fixed tissues, even after sectioning, extraction with detergents and several washes.ref

Biotinylated Dextrans

In addition to the low molecular weight biotinylated tracers described above, we prepare a variety of biotinylated dextrans (Figure 4.3.2, Molecular Probes dextran conjugates—Table 14.4), including dextrans that are double-labeled with fluorophores and biotin moieties for correlated fluorescence and electron microscopy studies. We currently offer the following biotinylated dextrans:

  • Dextran, biotin, 3000 MW, lysine fixable (BDA-3000) (D7135)
  • Dextran, biotin, 10,000 MW, lysine fixable (BDA-10,000) (D1956)
  • Dextran, biotin, 70,000 MW, lysine fixable (BDA-70,000) (D1957)
  • Dextran, biotin, 500,000 MW, lysine fixable (BDA-500,000) (D7142)
  • Dextran, fluorescein and biotin, 3000 MW, anionic, lysine fixable (micro-emerald) (D7156)
  • Dextran, fluorescein and biotin, 10,000 MW, anionic, lysine fixable (mini-emerald) (D7178)
  • Dextran, tetramethylrhodamine and biotin, 3000 MW, lysine fixable (micro-ruby) (D7162)
  • Dextran, tetramethylrhodamine and biotin, 10,000 MW, lysine fixable (mini-ruby) (D3312)

Dextrans are hydrophilic polysaccharides characterized by their moderate to high molecular weight, good water solubility and low toxicity. They are biologically inert due to their uncommon poly-(α-D-1,6-glucose) linkages, which render them resistant to cleavage by most endogenous cellular glycosidases. Dextrans are widely used as both anterograde and retrograde tracers in neurons and for many other diverse applications; see Fluorescent and Biotinylated Dextrans—Section 14.5 for a discussion of the applications of these reagents, particularly as cell tracers.

Motor neuron in a three-day chick embryo  
Figure 4.3.2
Motor neuron in a three-day chick embryo labeled with lysine-fixable, biotinylated 3000 MW dextran (BDA-3000, D7135). Filled neurons were detected with biotinylated horseradish peroxidase (P917) and diaminobenzidine using standard avidin/streptavidin bridging techniques. Reprinted with permission from ref.

Biotinylated and DSB-X Biotin–Labeled Proteins

Our biotinylated primary and secondary antibodies, F(ab')2 fragments, phycobiliproteins and enzymes are reliable detection reagents for a broad assortment of assays; for more information, see Ultrasensitive Detection Technology—Chapter 6 and Antibodies, Avidins and Lectins—Chapter 7. Biotinylated R-phycoerythrin (P811) and biotinylated horseradish peroxidase (P917) can be used in combination with an avidin or streptavidin bridge to amplify the detection of biotinylated targets.

Biotinylated Primary Antibodies

We prepare biotin conjugates of several primary antibodies, including:

  • Anti-bromodeoxyuridine, mouse IgG1, monoclonal MoBU-1, biotin conjugate (biotinylated anti-BrdU, B35138)
  • Anti–HuC/HuD neuronal protein (human), mouse IgG2b, monoclonal 16A11, biotin-XX conjugate (A21272)
  • Anti–α-tubulin (bovine), mouse IgG1, monoclonal 236-10501, biotin-XX conjugate (A21371)
  • Anti–dinitrophenyl-KLH, rabbit IgG fraction, biotin-XX conjugate (A6435)
  • Anti–fluorescein/Oregon Green, rabbit IgG fraction, biotin-XX conjugate (A982)
  • Anti–lucifer yellow, rabbit IgG fraction, biotin-XX conjugate (A5751)
  • Anti–green-fluorescent protein (GFP), rabbit IgG fraction, biotin-XX conjugate (A10259)
  • Anti–green-fluorescent protein (GFP), chicken IgY fraction, biotin-XX conjugate (A10263)

For a complete list of primary antibodies available from Invitrogen, go to www.invitrogen.com/handbook/antibodies.

Biotinylated and DSB-X Biotin–Labeled Secondary Antibodies

We prepare biotin and DSB-X biotin conjugates of several commonly used secondary antibodies, including:

  • Biotin-XX F(ab')2 fragment of goat anti–mouse IgG (H+L) (B11027)
  • Biotin-XX F(ab')2 fragment of goat anti–rabbit IgG (H+L) (B21078)
  • Biotin-XX goat anti–mouse IgG (H+L) (B2763)
  • Biotin-XX goat anti–mouse IgG, IgA, IgM (H+L) (A10676)
  • Biotin-XX goat anti–mouse IgG1 (γ1) (A10519)
  • Biotin-XX goat anti–rabbit IgG (H+L) (B2770)
  • Biotin-XX goat anti–rat IgG (H+L) (A10517)
  • Biotin-XX mouse anti–human IgG1 (A10650)
  • Biotin-XX mouse anti–human IgG4 (A10663)
  • Biotin-XX rabbit anti–goat IgG (H+L) (A10518)
  • DSB-X biotin goat anti–mouse IgG (H+L) (D20691)
  • DSB-X biotin goat anti–mouse IgM (µ chain) (D20693)
  • DSB-X biotin goat anti–rat IgG (H+L) (D20697)
  • DSB-X biotin goat anti–chicken IgG (H+L) (D20701)
  • DSB-X biotin donkey anti–goat IgG (H+L) (D20698)

Targets complexed with DSB-X biotin–labeled antibodies can be selectively detected with avidin or streptavidin conjugates or isolated on affinity matrices, including streptavidin agarose (S951, D20658; Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6), and then rapidly released with D-biotin (B1595, B20656; Biotinylation and Haptenylation Reagents—Section 4.2) under extremely gentle conditions ref (Figure 4.3.3). See Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6 for a complete description of our unique DSB-X biotin technology.

Streptavidin Agarose  
Figure 4.3.3
Diagram illustrating the use of streptavidin agarose and a DSB-X biotin bioconjugate in affinity chromatography. A DSB-X biotin–labeled IgG antibody and its target antigen are used as an example.


BioGEE: A Biotinylated Glutathione Analog

Biotinylated glutathione ethyl ester (BioGEE, G36000; structure) is a cell-permeant, biotinylated glutathione analog for detecting glutathiolation. Under conditions of oxidative stress, cells may transiently incorporate glutathione into proteins. Stressed cells incubated with BioGEE will also incorporate this biotinylated glutathione derivative into proteins, facilitating the identification of oxidation-sensitive proteins.ref Once these cells are fixed and permeabilized, glutathiolation levels can be detected with a fluorescent streptavidin conjugate (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6, Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9) using either flow cytometry or fluorescence microscopy. Proteins glutathiolated with BioGEE can be captured using streptavidin agarose (S951, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6) and analyzed by mass spectrometry or by western blotting methods.ref

Biotinylated Microspheres

Biotinylated FluoSpheres polystyrene microspheres have significant potential for signal amplification techniques, as described in Microspheres—Section 6.5. Like biotinylated R-phycoerythrin (P811, see above), they can be used with bridging techniques to detect biotinylated targets. We currently offer the following biotinylated FluoSpheres microspheres:

  • FluoSpheres biotin-labeled microspheres, 0.04 µm, yellow-green fluorescent (505/515) *1% solids* (F8766)
  • FluoSpheres biotin-labeled microspheres, 0.2 µm, yellow-green fluorescent (505/515) *1% solids* (F8767)
  • FluoSpheres biotin-labeled microspheres, 1.0 µm, yellow-green fluorescent (505/515) *1% solids* (F8768)
  • FluoSpheres biotin-labeled microspheres, 1.0 µm, nonfluorescent *1% solids* (F8769)

FluoSpheres polystyrene microspheres satisfy several prerequisites of ideal long-term biological tracers. Because the dyes in our microspheres are incorporated throughout the microsphere rather than just on its surface, the fluorescence output per microsphere is significantly greater than that obtained from protein or dextran conjugates and is relatively immune to photobleaching and other environment-dependent effects. FluoSpheres microspheres are also biologically inert and physically durable, and are available with a large number of uniform sizes and surface properties.

Biotinylated Qdot Nanocrystals

The Qdot 605 and Qdot 655 Biotin Conjugate Kits (Q10301MP, Q10321MP) provide Qdot nanocrystals that have been functionalized with biotin on their surface via carbodiimide-mediated coupling. The biotinylated Qdot 605 or biotinylated Qdot 655 conjugate typically incorporates 5–7 biotin groups per Qdot nanocrystal. In addition to biotinylated Qdot nanocrystals, each kit also provides Qdot Incubation Buffer, which is formulated specifically to achieve improved signal-to-noise ratios in immunohistochemical applications. Biotinylated Qdot nanocrystals have been used alongside biotinylated microspheres (see above) for analysis of the effects of fluorescent label size on ligand–receptor binding dynamics and equilibrium by fluorescence correlation spectroscopy ref (Fluorescence Correlation Spectroscopy (FCS)—Note 1.3).

Biotinylated Nucleotides

Biotinylated nucleic acids are common nonisotopic probes used in hybridization techniques. Nucleoside triphosphate analogs such as our ChromaTide UTP and dUTP nucleotides and aha-dUTP and aha-dCTP nucleotides (Labeling Oligonucleotides and Nucleic Acids—Section 8.2) are important reagents for preparing labeled nucleic acids for use as hybridization probes. The biotinylated aminohexylacrylamido-dUTP (biotin aha-dUTP) and biotinylated aminohexylacrylamido-dCTP (biotin aha-dCTP) derivatives (B32766, structure; B32772) each contain a long 11-atom spacer between the biotin and its attachment point on the nucleic acid to facilitate its detection and signal amplification by fluorophore and enzyme conjugates of avidin and streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6, Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9).

Biotinylated Site-Selective Probes

Biotin conjugates of moderately low molecular weight ligands provide a means of amplifying the detection of ligand binding using fluorophore- or enzyme-labeled avidins or streptavidins. They may also be useful for immobilizing receptor ligands on streptavidin agarose (S951, D20658) or CaptAvidin agarose (C21386, Figure 4.3.4) for affinity isolation of receptors. See Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6 for a description of these avidin conjugates, as well as other affinity isolation methods that use biotin- or DSB-X biotin–labeled reagents.

Our biotinylated ligands include the:

CaptAvidin agarose  

 

Figure 4.3.4 Diagram of the use of CaptAvidin agarose (C21386) in affinity chromatography. A biotinylated IgG molecule and target antigen are used as an example.

Biotinylated Lipids

Our extensive selection of labeled phospholipids includes two phospholipid derivatives of biotin and biotin-X:

  • Biotin DHPE (N-(biotinoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (B1550, structure)
  • Biotin-X DHPE (N-((6-(biotinoyl)amino)hexanoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (B1616, structure)

These biotinylated lipids, which are also described in Chemical Crosslinking Reagents—Section 5.2, can be used to prepare liposomes that retain high affinity for avidin conjugates and can be captured for downstream analysis using streptavidin agarose ref (S951, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6). More information on our labeled fatty acids and phospholipids can be found in Fatty Acid Analogs and Phospholipids—Section 13.2.

Data Table

Cat # Links MW Storage Soluble Abs EC Em Solvent Notes
A12922 icon 1141.31 D,L DMSO, H2O 591 80,000 618 pH 7  
A12923 icon 1209.66 D,L DMSO, H2O 556 99,000 572 pH 7  
A12924 icon 974.98 D,L DMSO, H2O 494 62,000 520 pH 7  
B1196   ~8400 F,D H2O <300   none   1
B1370 icon 831.01 L DMF, pH >6 494 75,000 518 pH 9 2
B1550 icon 1019.45 FF,D see Notes <300   none   3
B1616 icon 1132.61 FF,D see Notes <300   none   3
B7474 icon ~1300 F MeOH, H2O <300   none   1, 4
B10570 icon 644.70 L DMSO 494 68,000 523 pH 9 2
B32766 icon 1041.78 FF H2O <300   none   5
B32772   ~1050 FF H2O <300   none   5
E3477   ~6600 FF,D H2O <300   none   1
E3480 icon see Notes FF,D,L H2O 596 ND 612 pH 7 6, 7
E13345 icon see Notes FF,D,L H2O 497 ND 520 pH 8 6, 8
E35350 icon see Notes FF,D,L H2O 554 ND 568 pH 7 6, 9
E35351 icon see Notes FF,D,L H2O 653 ND 671 pH 7 6, 10
G36000 icon 561.67 F,D DMSO <300   none    
L2601 icon icon 873.10 D,L H2O 428 11,000 531 H2O  
L6950 icon icon 850.03 D,L H2O 428 11,000 532 pH 7  
O12920 icon 887.39 L DMSO, H2O 495 66,000 522 pH 9 11
T12921 icon 869.09 D,L DMSO 554 103,000 581 pH 7  
  1. α-Bungarotoxin, EGF and phallotoxin conjugates have approximately 1 label per peptide.
  2. Absorption and fluorescence of fluorescein derivatives are pH dependent. Extinction coefficients and fluorescence quantum yields decrease markedly at pH <7.
  3. Chloroform is the most generally useful solvent for preparing stock solutions of phospholipids (including sphingomyelins). Glycerophosphocholines are usually freely soluble in ethanol. Most other glycerophospholipids (phosphoethanolamines, phosphatidic acids and phosphoglycerols) are less soluble in ethanol, but solutions up to 1–2 mg/mL should be obtainable, using sonication to aid dispersion if necessary. Labeling of cells with fluorescent phospholipids can be enhanced by addition of cyclodextrins during incubation.ref
  4. Although this phallotoxin is water soluble, storage in water is not recommended, particularly in dilute solution.
  5. This product is supplied as a ready-made solution in the solvent indicated under "Soluble."
  6. ND = not determined.
  7. E3480 is a complex of E3477 with Texas Red streptavidin, which typically incorporates 3 dyes/streptavidin (MW ~52,800).
  8. E13345 is a complex of E3477 with Alexa Fluor 488 streptavidin, which typically incorporates 5 dyes/streptavidin (MW ~52,800).
  9. E35350 is a complex of E3477 with Alexa Fluor 555 streptavidin, which typically incorporates 3 dyes/streptavidin (MW ~52,800).
  10. E35351 is a complex of E3477 with Alexa Fluor 647 streptavidin, which typically incorporates 3 dyes/streptavidin (MW ~52,800).
  11. Absorption and fluorescence of Oregon Green 488 derivatives are pH dependent only in moderately acidic solutions (pH <5).
For Research Use Only. Not for use in diagnostic procedures.