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.
Fluorescein biotin (B1370, ) was developed as an alternative to radioactive biotin for detecting and quantitating biotin-binding sites by either fluorescence or absorbance. A fluorescence polarization–based assay that employs competitive binding of fluorescein biotin to assess the degree of protein biotinylation has been reported (Fluorescence Polarization (FP)—Note 1.4). A similar derivative was used for determining avidin and biotin concentrations by fluorescence depolarization.
Our biotin-4-fluorescein (B10570, ) 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. The strong quenching associated with avidin binding to biotin-4-fluorescein can be used to accurately measure the concentration of avidin or streptavidin (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.
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:
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 (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.
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.
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 .
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 (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.
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; ) 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. 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 7.23) 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.
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.
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 (Fluorescence Correlation Spectroscopy (FCS)—Note 1.3).
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, ; 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 7.23).
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:
- Biotin-X conjugate of annexin V (A13204, Assays for Apoptosis—Section 15.5), for detecting the externalization of phosphatidylserine, an early indicator of apoptosis
- Biotin-XX conjugate of α-bungarotoxin (B1196, Probes for Neurotransmitter Receptors—Section 16.2), for labeling the nicotinic acetylcholine receptor (nAChR)
- Biotin-XX cholera toxin subunit B (C34779, Lectins and Other Carbohydrate-Binding Proteins—Section 7.7), which binds to cell-surface ganglioside GM1 receptors and is a marker of lipid rafts
- Biotin-XX conjugate of epidermal growth factor (EGF, E3477), as well as biotinylated EGF complexed with Alexa Fluor 488 streptavidin (E13345, ), Alexa Fluor 555 streptavidin (E35350), Alexa Fluor 647 streptavidin (E35351) or Texas Red streptavidin (E3480, ), for labeling EGF receptors (Probes for Following Receptor Binding and Phagocytosis—Section 16.1)
- Biotin-XX conjugate of isolectin IB4 from Griffonia simplicifolia (I21414, Lectins and Other Carbohydrate-Binding Proteins—Section 7.7), for detecting α-galactosyl moieties in microglia and other glycoproteins
- Biotin-XX conjugate of phalloidin (B7474, Probes for Actin—Section 11.1), for labeling F-actin
- Biotin-XX conjugate of transferrin (T23363, Probes for Following Receptor Binding and Phagocytosis—Section 16.1), for following intracellular trafficking of this iron-carrying protein
- Biotin-XX tyramide, which is an amplification reagent used in several of our TSA Kits (TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2, Tyramide Signal Amplification (TSA) Kits—Table 6.1) for high-sensitivity detection of targets in cells and tissues
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.
Our extensive selection of labeled phospholipids includes two phospholipid derivatives of biotin and biotin-X:
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 (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.
|A12922||1141.31||D,L||DMSO, H2O||591||80,000||618||pH 7|
|A12923||1209.66||D,L||DMSO, H2O||556||99,000||572||pH 7|
|A12924||974.98||D,L||DMSO, H2O||494||62,000||520||pH 7|
|B1370||831.01||L||DMF, pH >6||494||75,000||518||pH 9||2|
|B7474||~1300||F||MeOH, H2O||<300||none||1, 4|
|E3480||see Notes||FF,D,L||H2O||596||ND||612||pH 7||6, 7|
|E13345||see Notes||FF,D,L||H2O||497||ND||520||pH 8||6, 8|
|E35350||see Notes||FF,D,L||H2O||554||ND||568||pH 7||6, 9|
|E35351||see Notes||FF,D,L||H2O||653||ND||671||pH 7||6, 10|
|O12920||887.39||L||DMSO, H2O||495||66,000||522||pH 9||11|