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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. |
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:
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 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 can be used in combination with an avidin or streptavidin bridge to amplify the detection of biotinylated targets.
We prepare biotin conjugates of several primary antibodies, including:
For a complete list of primary antibodies available from Invitrogen, go to www.invitrogen.com/handbook/antibodies.
We prepare biotin and DSB-X biotin conjugates of several commonly used secondary antibodies, including:
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, 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. |
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 7.9) 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.
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 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 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).
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) 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:
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. |
In addition to fluorescently labeled phospholipids, two phospholipid derivatives of biotin and biotin-X have proven useful:
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.
For a detailed explanation of column headings, see Definitions of Data Table Contents
Cat # | MW | Storage | Soluble | Abs | EC | Em | Solvent | Notes |
---|---|---|---|---|---|---|---|---|
A12922 Alexa Fluor 594 biocytin | 1141.31 | D,L | DMSO, H2O | 591 | 80,000 | 618 | pH 7 | |
Alexa Fluor 546 biocytin | 1209.66 | D,L | DMSO, H2O | 556 | 99,000 | 572 | pH 7 | |
A12924 Alexa Fluor 488 biocytin | 974.98 | D,L | DMSO, H2O | 494 | 62,000 | 520 | pH 7 | |
B1196 α-bungarotoxin, biotin-XX | ~8400 | F,D | H2O | <300 | none | 1 | ||
B1370 fluorescein biotin | 831.01 | L | DMF, pH >6 | 494 | 75,000 | 518 | pH 9 | 2 |
biotin DHPE | 1019.45 | FF,D | see Notes | <300 | none | 3 | ||
biotin-X DHPE | 1132.61 | FF,D | see Notes | <300 | none | 3 | ||
B7474 biotin-XX phalloidin | ~1300 | F | MeOH, H2O | <300 | none | 1, 4 | ||
B10570 biotin-4-fluorescein | 644.70 | L | DMSO | 494 | 68,000 | 523 | pH 9 | 2 |
biotin-aha-dUTP | 1041.78 | FF | H2O | <300 | none | 5 | ||
biotin-aha-dCTP | ~1050 | FF | H2O | <300 | none | 5 | ||
E3477 epidermal growth factor, biotin-XX | ~6600 | FF,D | H2O | <300 | none | 1 | ||
E3480 epidermal growth factor, Texas Red complex | see Notes | FF,D,L | H2O | 596 | ND | 612 | pH 7 | 6, 7 |
E13345 epidermal growth factor, Alexa Fluor 488 complex | see Notes | FF,D,L | H2O | 497 | ND | 520 | pH 8 | 6, 8 |
E35350 epidermal growth factor, Alexa Fluor 555 complex | see Notes | FF,D,L | H2O | 554 | ND | 568 | pH 7 | 6, 9 |
E35351 epidermal growth factor, Alexa Fluor 647 complex | see Notes | FF,D,L | H2O | 653 | ND | 671 | pH 7 | 6, 10 |
G36000 BioGEE | 561.67 | F,D | DMSO | <300 | none | |||
lucifer yellow cadaverine biotin-X | 873.10 | D,L | H2O | 428 | 11,000 | 531 | H2O | |
lucifer yellow biocytin | 850.03 | D,L | H2O | 428 | 11,000 | 532 | pH 7 | |
Oregon Green 488 biocytin | 887.39 | L | DMSO, H2O | 495 | 66,000 | 522 | pH 9 | 11 |
T12921 biocytin TMR | 869.09 | D,L | DMSO | 554 | 103,000 | 581 | pH 7 | |
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For Research Use Only. Not for use in diagnostic procedures.