Kits for Labeling Proteins and Nucleic Acids—Section 1.2

We provide a vast number of stand-alone reagents for preparing bioconjugates, most of which are described in detail in other sections of this chapter. This section describes the many specialized kits that we have developed for covalently labeling proteins and nucleic acids with our organic dyes and haptens (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3).

As an alternative to direct conjugation of primary antibodies with our reactive dyes and haptens, we recommend using Zenon technology (Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3) to form labeled antibody complexes. Zenon labeling can be completed in minutes in quantitative yield starting with as little as submicrogram quantities of antibody, and the conjugate brightness can easily be adjusted by modifying the stoichiometry of the reagents. Although they do not provide covalent labeling, the Zenon Antibody Labeling Kits are listed in Active esters and kits for labeling proteins and nucleic acids—Table 1.2, as well as in Zenon Labeling Kits—Table 7.14.

APEX Antibody Labeling Kits

APEX Antibody Labeling Kits (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3) provide a convenient method for covalently labeling small amounts (10–20 µg) of IgG antibody with Alexa Fluor dyes, Oregon Green 488 dye or Pacific Blue dye. A primary antibody directly labeled with a fluorophore often produces lower background fluorescence and less nonspecific binding than secondary antibodies. Furthermore, multiple primary antibodies of the same isotype or derived from the same species can easily be used in the same immunostaining experiment if they are directly labeled with compatible fluorophores. Many IgG antibodies, however, are often only available in small quantities and packaged with stabilizing proteins, such as BSA, or other contaminants that can interfere with the amine-reactive labeling reagents.

APEX Antibody Labeling Kits are specifically designed to allow labeling of small amounts of IgG antibody, even in the presence of contaminants. These kits utilize a solid-phase labeling technique that captures the IgG antibody on resin inside an APEX antibody labeling tip (Figure 1.122). Any contaminants, including stabilizing proteins or amine-containing buffers, are eluted through the tip before labeling. After applying the amine-reactive fluorophore to the IgG antibody trapped on the resin, a fluorescent IgG conjugate is formed and subsequently eluted from the resin using elution buffer. The fluorescent IgG conjugate is ready to use in an imaging or flow cytometry assay in as little as 2.5 hours with minimal hands-on time. The typical yield of labeled antibody using this method is between 40 and 80%.

Each APEX Antibody Labeling Kit provides all reagents required to perform five separate labeling reactions of 10–20 µg IgG antibody, including:

• Five vials of amine-reactive fluorescent label
• Five APEX antibody labeling tips
• Wash, labeling, neutralization and elution buffers
• Dimethylsulfoxide (DMSO)
• Labeling protocol (APEX Antibody Labeling Kits)

For labeling larger amounts of protein, we recommend the Alexa Fluor Microscale Protein Labeling Kits, which are optimized for 20–100 µg samples of proteins between 10,000 and 150,000 daltons; the Alexa Fluor Monoclonal Antibody Labeling Kits, which are optimized for 100 µg samples of mono- or polyclonal antibodies; or the Alexa Fluor Protein Labeling Kits, which are optimized for 1 mg samples of >30,000-dalton proteins.

Alexa Fluor Microscale Protein Labeling Kits

Alexa Fluor Microscale Protein Labeling Kits (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3) provide a convenient means for labeling small amounts (20–100 µg) of purified protein with the highly fluorescent Alexa Fluor dyes. Spin columns are used to purify the labeled protein, with yields between 60 and 90% depending primarily on the molecular weight of the starting material. Labeling and purification can be completed in as little as 30 minutes.

These kits have been optimized for labeling proteins with molecular weights between 12,000 and 150,000 daltons and contain everything needed to perform three labeling reactions and to separate the resulting conjugates from excess dye. Each Alexa Fluor Microscale Protein Labeling Kit provides:

• Three vials of the succinimidyl ester (or tetrafluorophenyl ester) of the corresponding fluorescent dye
• Sodium bicarbonate
• Reaction tubes
• Spin filters and purification resin
• ElutaTube microdialysis vials
• Detailed protocols for conjugation, purification and determination of the degree of labeling (Alexa Fluor Microscale Protein Labeling Kits)

Monoclonal Antibody Labeling Kits

Monoclonal Antibody Labeling Kits (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3) provide researchers with a simple yet efficient means of labeling small amounts of IgG antibodies with Alexa Fluor dyes (Figure 1.6). Unlike polyclonal antibodies and most other commercially available proteins, monoclonal antibodies are typically only available in small quantities. These kits contain everything needed to perform five separate labeling reactions containing ~100 µg each of carrier-free monoclonal IgG samples (although other proteins can be labeled).

Each Monoclonal Antibody Labeling Kit provides:

• Five vials of the succinimidyl ester (or tetrafluorophenyl ester) of the corresponding fluorescent dye
• Sodium bicarbonate
• Spin columns and collection tubes
• Detailed protocols for conjugation, purification and determination of the degree of labeling (Monoclonal Antibody Labeling Kits)

Simply dissolve the carrier-free monoclonal antibody at ~1 mg/mL in the provided buffer, then add it to one of the five vials of amine-reactive dye; no organic solvents are required. Purification of the fluorescent conjugate is accomplished on a size-exclusion spin column optimized for proteins with molecular weight greater than 30,000 daltons. Labeling and purification can be completed in less than two hours.

SAIVI Antibody Labeling Kits

The optimal fluorescent antibody conjugate for in vitro detection assays produces an intense fluorescent signal yet retains the binding selectivity and kinetics of the unlabeled antibody. When preparing a fluorescent antibody conjugate for in vivo animal imaging, however, the pharmacokinetics of the labeled probe must also be considered. In both in vitro and in vivo applications, a more heavily labeled antibody does not usually produce a better conjugate. Antibody conjugates with a very high degree of labeling (DOL) typically precipitate out of solution, bind nonspecifically or exhibit intramolecular fluorescence quenching, making it necessary to have a less-than-maximal DOL to obtain a functional fluorescent antibody. For in vivo labeling experiments, the DOL of an antibody conjugate is even more severely restricted because it has significant consequences for the biodistribution and clearance of the probe. These additional constraints have led to the development of the SAIVI Antibody Labeling Kits for small animal in vivo imaging applications (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3).

SAIVI Antibody Labeling Kits feature reactive far-red and near-infrared Alexa Fluor dyes, along with a labeling protocol specifically designed to produce a suitable DOL for in vivo imaging applications. When optimally labeled, a fluorescent antibody conjugate produces an intense, targeted fluorescent signal that persists throughout the in vivo study, without significant redistribution or clearance of the probe.

The conjugation protocol in the SAIVI Rapid Antibody Labeling Kits produces an optimal DOL (~2 fluorophores per antibody) over a sixfold antibody concentration range with no adjustments in reaction volume, dye concentration or antibody concentration necessary. Purification of the dye-labeled antibody is achieved usng a simple protocol that can be completed in less than 10 minutes. With these kits, optimally labeled antibodies are ready for applications that require azide-free reagents, such as live-cell imaging or direct injections into animals. SAIVI Rapid Antibody Labeling Kits containing either Alexa Fluor 680 or Alexa Fluor 790 dye (S30045, S30046) provide sufficient reagents for three labeling reactions of 0.5–3 mg of protein each, including:

• Three vials of amine-reactive Alexa Fluor 680 or Alexa Fluor 790 dye
• Sodium bicarbonate
• Regulator solution
• Purification columns and purification resin
• Phosphate-buffered saline (PBS)
• Syringes, syringe filters, column-loading pipettes and catch tubes
• Detailed protocols for conjugation, purification and determination of the degree of labeling (SAIVI Rapid Antibody Labeling Kits)

To control the DOL of the antibody conjugate, the SAIVI Alexa Fluor 647 Antibody/Protein Labeling Kit (S30044) includes a DOL modulating reagent and instructions for decreasing the DOL from its intrinsic highest value by adding specific amounts of this reagent to the labeling reaction. With this method, antibody preparations with varying ratios of dye to protein can be quickly and reproducibly obtained without significant alteration of labeling or purification conditions, allowing more efficient optimization for in vivo imaging applications. Each kit provides sufficient reagents for three labeling reactions of 1 mg protein each, including:

• Three vials of amine-reactive Alexa Fluor 647 dye
• Sodium bicarbonate
• DOL modulating reagent
• Purification columns and purification resin
• Wash buffer
• Column funnels, column holders, disposable pipettes and collection tubes
• Detailed protocols for conjugation, purification and determination of the degree of labeling (SAIVI Alexa Fluor 647 Antibody/Protein Labeling Kit)

Easy-to-Use Protein Labeling Kits

Our easy-to-use Protein Labeling Kits (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3) provide a nearly effortless way to label proteins, especially IgG antibodies, with a fluorescent dye (Figure 1.5) Simply add ~1 mg of protein (in a volume of ~500 µL and free of amine-containing buffers such as Tris) to one of the three included vials, which contain a premeasured quantity of amine-reactive dye and a magnetic stir bar. Because the reactive dyes used in these kits are water soluble, no organic solvents are required. Purification is accomplished on a gravity-feed size-exclusion column, which is supplied with the kit. Labeling and purification can be completed in about two hours, with very little hands-on time.

Each Protein Labeling Kit provides sufficient reagents for labeling three ~1 mg protein samples of a 150,000-dalton protein (such as an IgG), including:

• Three vials of the succinimidyl ester (or tetrafluorophenyl ester) of the corresponding fluorescent dye, each containing a magnetic stir bar
• Sodium bicarbonate
• Gravity-feed columns, a size-exclusion resin and concentrated elution buffer
• Column funnels, foam column holders, disposable pipettes and collection tubes
• Detailed protocols for conjugation, purification and determination of the degree of labeling (Protein Labeling Kits)

Researchers have modified α-synuclein using either the Oregon Green 488 Protein Labeling Kit (O10241) or the Alexa Fluor 594 Protein Labeling Kit (A10239). They intentionally produced fluorescent conjugates with a low degree of substitution (DOS) so as not to perturb oligomerization. When comparing Oregon Green 488 synuclein with Alexa Fluor 594 synuclein, they observed essentially the same results in terms of fibril formation as analyzed by fluorescence polarization. They also detected the Oregon Green 488 synuclein conjugate with mouse monoclonal anti-fluorescein/Oregon Green antibody (monoclonal 4-4-20, A6421) and gold-labeled anti–mouse IgG antibody using electron microscopy.

FluoReporter Protein Labeling Kits

The FluoReporter Protein Labeling Kits (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3) facilitate research-scale preparation of protein conjugates labeled with some of our brightest fluorescent dyes. Typically, labeling and purifying conjugates with the FluoReporter Protein Labeling Kits can be completed in under three hours, with very little hands-on time. First, the amount of dye necessary for the desired protein sample is calculated using the guidelines outlined in the kit protocol. After dissolving the dye in dimethylsulfoxide (DMSO), the calculated amount of dye is added to the protein and the reaction is incubated for 1–1.5 hours. Purification is easily accomplished using convenient spin columns designed for use with proteins of molecular weight greater than 30,000 daltons.

Each FluoReporter Protein Labeling Kits provides sufficient reagents to label 5 to 10 protein samples of 0.2–2 mg each in 200 µL volumes :

• Five vials of the amine-reactive dye
• Dimethylsulfoxide (DMSO)
• Reaction tubes, each containing a stir bar
• Spin columns and collection tubes
• Detailed protocols for conjugation, purification and determination of the degree of labeling (FluoReporter Protein Labeling Kits)

Zenon Antibody Labeling Kits

The Zenon Antibody Labeling Kits (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Zenon Labeling Kits—Table 7.14) are useful for the rapid and quantitative labeling of antibodies with dyes (including phycobiliproteins and their tandem conjugates), biotin or enzymes (Figure 7.56). Zenon Antibody Labeling Kits are designed to label intact antibodies in amounts from less than 1 µg to as much as 50 µg, starting with a purified antibody fraction or with a crude antibody preparation such as serum, ascites fluid or a hybridoma supernatant. The Zenon antibody labeling technology is discussed further in Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3.

Biotin-XX Microscale Protein Labeling Kits

The Biotin-XX Microscale Protein Labeling Kit (B30010; Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3) provides a convenient means for biotinylating small amounts (20–100 µg) of purified protein. The water-soluble biotin-XX sulfosuccinimidyl ester has a 14-atom spacer () that enhances the binding of biotin derivatives to avidin's relatively deep binding sites. Spin columns are used to purify the labeled protein, with yields between 60 and 90% depending primarily on the molecular weight of the starting material. Labeling and purification can be completed in as little as 30 minutes.

This kit, which has been optimized for labeling proteins with molecular weights between 12,000 and 150,000 daltons, contains everything needed to perform three labeling reactions and to separate the resulting conjugates from excess reactive biotin. Each Biotin-XX Microscale Protein Labeling Kit provides:

• Three vials of biotin-XX, sulfosuccinimidyl ester
• Sodium bicarbonate
• Reaction tubes
• Spin filters and purification resin
• ElutaTube microdialysis vials
• Detailed protocols for conjugation and purification (Biotin-XX Microscale Protein Labeling Kit)

For determining the degree of labeling, the FluoReporter Biotin Quantitation Assay Kit for proteins is available separately (F30751) or in combination with the Biotin-XX Microscale Protein Labeling Kit (B30756). When biotinylating larger amounts of protein, we recommend the FluoReporter Biotin-XX Protein Labeling Kit, which is optimized for 5–20 mg samples, or the FluoReporter Mini-Biotin-XX Protein Labeling Kit, which is optimized for 0.1–3 mg samples of >30,000-dalton proteins.

FluoReporter Mini-Biotin-XX Protein Labeling Kit

The FluoReporter Mini-Biotin-XX Protein Labeling Kit (F6347; Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3) permits efficient biotinylation of small amounts of antibodies or other proteins. The water-soluble biotin-XX sulfosuccinimidyl ester has a 14-atom spacer () that enhances the binding of biotin derivatives to avidin's relatively deep binding sites. The ready-to-use spin columns provide a convenient method of purifying the biotinylated protein from excess reagents.

Each FluoReporter Mini-Biotin-XX Protein Labeling Kit provides sufficient reagents for five labeling reactions of 0.1–3 mg protein each, including:

• Five vials of biotin-XX, sulfosuccinimidyl ester
• Reaction tubes, each containing a stir bar
• Spin columns and collection tubes
• Dialysis tubing
• Detailed protocols for conjugation and purification (FluoReporter Mini-Biotin-XX Protein Labeling Kit)

FluoReporter Biotin-XX Protein Labeling Kit

The FluoReporter Biotin-XX Protein Labeling Kit (F2610; Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3) is designed to provide five biotinylation reactions, each containing 5–20 mg of protein. A gel filtration column is used to purify the labeled proteins from excess biotin reagent. Once purified, the degree of biotinylation can be determined using the included avidin–biotin displacement assay; biotinylated goat IgG is provided as a standard.

Each FluoReporter Biotin-XX Protein Labeling Kit provides sufficient reagents for five labeling reactions of 5–20 mg protein each, including:

• Biotin-XX, succinimidyl ester
• Dimethylsulfoxide(DMSO)
• Gel filtration column
• Avidin–HABA complex
• Biotinylated goat IgG
• Detailed protocols for conjugation, purification and determination of degree of labeling (FluoReporter Biotin-XX Protein Labeling Kit)

FluoReporter Biotin/DNP Protein Labeling Kit

The FluoReporter Biotin/DNP Protein Labeling Kit (F6348; Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3) provides the necessary reagents for labeling proteins with DNP-X–biocytin-X succinimidyl ester (). The degree of biotinylation of proteins labeled with this reagent can be assessed from the optical absorbance of DNP (ε = 15,000 cm^-1M^-1 at ~360 nm). The conjugates are recognized by both avidin derivatives (and anti-biotin antibodies) and by anti-DNP antibodies, permitting a choice of detection techniques.

Each FluoReporter Biotin/DNP Protein Labeling Kit contains sufficient reagents for 5 to 10 labeling reactions of 0.2–2 mg of protein each, including:

• DNP-X–biocytin-X, succinimidyl ester (five vials)
• Dimethylsulfoxide (DMSO) for dissolving the succinimidyl ester
• Reaction tubes
• Spin columns and collection tubes
• Detailed protocols for conjugation, purification and determination of the degree of labeling (FluoReporter Biotin/DNP Protein Labeling Kit)

DSB-X Biotin Protein Labeling Kit

Our unique DSB-X biotin technology, which is described in detail in Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6, permits the facile reversal of the biotin–avidin interaction under extremely gentle conditions. DSB-X biotin succinimidyl ester, a derivative of desthiobiotin (Figure 4.1) with an additional seven-atom spacer, reacts with amine groups of biomolecules to form stable amides. Like biotin conjugates, the DSB-X biotin conjugate can be detected with any avidin or streptavidin derivative; with DSB-X biotin conjugates, however, this binding is almost totally reversed by addition of free biotin (B1595, B20656; Biotinylation and Haptenylation Reagents—Section 4.2) at neutral pH and normal ionic strength.

The DSB-X Biotin Protein Labeling Kit (D20655; Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3) contains the reagents required for five protein conjugations of 0.5–3 mg each, including:

• DSB-X biotin, succinimidyl ester (five vials)
• Dimethylsulfoxide (DMSO) for dissolving the succinimidyl ester
• Reaction tubes
• Purification resin, spin columns and collection tubes for small-scale purifications
• Dialysis tubing for larger-scale separations
• Detailed protocols for conjugation and purification (DSB-X Biotin Protein Labeling Kit)

The DSB-X Bioconjugate Isolation Kit (D20658, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6) provide the reagents and protocols for affinity isolation of DSB-X biotin conjugates using streptavidin agarose.

ULYSIS Nucleic Acid Labeling Kits

The ULYSIS Alexa Fluor Nucleic Acid Labeling Kits (Labeling Oligonucleotides and Nucleic Acids—Section 8.2; Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3, Spectral characteristics of the fluorescent dyes available in the ULYSIS Nucleic Acid Labeling Kits—Table 8.8) provide a simple, fail-safe method for producing fluorescent hybridization probes by combining our Alexa Fluor fluorophores with the versatile, patented Universal Linkage System (ULS) platinum-based chemistry developed by KREATECH Biotechnology BV. The ULS technology is based on the use of a platinum dye complex that forms a stable adduct with the N-7 position of guanine and, to a lesser extent, adenine bases in DNA, RNA, peptide–nucleic acid conjugates (PNA) and oligonucleotides (Figure 8.42). This labeling reaction takes only 15 minutes, and separation of the labeled nucleic acids from the unreacted ULS complex can be accomplished through use of a simple spin-column procedure (Figure 8.43).

In addition to ULYSIS Alexa Fluor Nucleic Acid Labeling Kits, we offer the ULYSIS Oregon Green 488 Nucleic Acid Labeling Kit.  Each of these ULYSIS Kits provides sufficient reagents for 20 labelings of 1 µg DNA each, including:

• ULS labeling reagent and appropriate solvent
• Labeling buffer
• Deoxyribonuclease I (DNase I), for digesting DNA longer than 1000 base-pairs prior to labeling
• DNase I storage and reaction buffers
• Control DNA from calf thymus
• Nuclease-free H₂O
• Detailed protocols for preparing fluorescent DNA hybridization probes for chromosome in situ hybridization and dot-blot hybridization (ULYSIS Nucleic Acid Labeling Kits)

Probes labeled using the ULYSIS Kits are stable indefinitely and hybridize effectively to target DNA. The ULS method has been used to prepare labeled probes for dot, Southern and Northern blot analysis, RNA and DNA in situ hybridization, multicolor FISH, comparative genome hybridization (CGH) and microarray analysis.

ARES DNA Labeling Kits

The ARES DNA Labeling Kits (Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3, Spectral characteristics of the fluorescent dyes available in the ARES DNA Labeling Kits—Table 8.9) provide a versatile, two-step method for labeling DNA with several of our premier fluorescent dyes (Figure 8.45). In the first step, an amine-modified nucleotide, 5-(3-aminoallyl)-dUTP (), is incorporated into DNA using conventional enzymatic labeling methods. This step ensures relatively uniform labeling of the probe with primary amine groups. The aminoallyl dUTP substrate used in this reaction is taken up efficiently by reverse transcription or nick translation, for which we provide the protocols; other enzymatic methods are also likely to be compatible. In the second step, the amine-modified DNA is chemically labeled using an amine-reactive fluorescent dye. This chemical reaction varies little in its efficiency from dye to dye, so that it is possible to use any combination of the ARES Kits, with their broad selection of the brightest and most photostable dyes, and obtain consistent DNA labeling. The labeling protocols provided generally result in about one dye per 12–15 bases, which we have determined to be optimal for fluorescence in situ hybridization (FISH) and dot-blot hybridization.

Each ARES DNA Labeling Kit provides sufficient reagents for 5 to 10 labelings of 1–5 µg DNA each, including:

• 5-(3-Aminoallyl)-dUTP
• Amine-reactive fluorescent dye and appropriate solvent
• Sodium bicarbonate
• Nuclease-free H₂O
• Detailed protocol for labeling DNA using reverse transcriptase or nick translation (ARES DNA Labeling Kits)

See Labeling Oligonucleotides and Nucleic Acids—Section 8.2 for a complete description of the ARES Kits and Detecting Nucleic Acid Hybridization—Section 8.5 for applications of nucleic acid probes prepared using the ARES reagents.

Alexa Fluor Oligonucleotide Amine Labeling Kits

The Alexa Fluor Oligonucleotide Amine Labeling Kits (Labeling Oligonucleotides and Nucleic Acids—Section 8.2; Active esters and kits for labeling proteins and nucleic acids—Table 1.2, Molecular Probes kits for protein and nucleic acid labeling—Table 1.3, Oligonucleotide Amine Labeling Kits—Table 8.10) provide the reagents required for labeling synthetic oligonucleotides that have amine groups incorporated at their 5'-terminus. Following purification by standard chromatographic or electrophoretic procedures, these singly labeled oligonucleotides can serve as primers for a variety of applications. The dye-labeled oligonucleotides may also serve as either fluorescence resonance energy transfer (FRET) acceptors or donors in hybridization reactions (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2).

Each Alexa Fluor Oligonucleotide Amine Labeling Kit contains sufficient reagents for three labelings of 50 µg each of an amine-modified oligonucleotide, including:

• Three vials of the amine-reactive dye
• Dimethylsulfoxide (DMSO)
• Three vials of labeling buffer
• Labeling protocol (Alexa Fluor Oligonucleotide Amine Labeling Kits)

FluoReporter Biotin Quantitation Assay Kit for Biotinylated Proteins

The FluoReporter Biotin Quantitation Assay Kit for biotinylated proteins (F30751) provides a sensitive fluorometric assay for accurately determining the number of biotin labels on a protein. This assay is based on the displacement of a ligand tagged with a quencher dye from the biotin binding sites of Biotective Green reagent. The FluoReporter biotin quantitation assay can detect from 4 to 80 picomoles of biotin in a sample, providing a 50-fold higher sensitivity than the HABA biotin binding assay described by Green. Furthermore, unlike the HABA biotin binding assay, which requires ~1 mg of protein sample, the FluoReporter biotin quantitation assay requires a minimum of 600 ng of a singly biotinylated IgG with molecular weight 150,000 daltons. For proteins of lower molecular weight or multiple biotin labels, less protein can be used. To expose any biotin groups in a multiply labeled protein that are sterically restricted and inaccessible to the Biotective Green reagent, this kit includes protease and an optional protocol for digesting the protein. With this preliminary digestion, biotin assay values agree well with MALDI-TOF determinations. The signal window of this assay has a Z' factor of 0.93. With excitation/emission maxima of 495/519 nm, this assay is compatible with any fluorescence-based microplate reader capable of detecting fluorescein (FITC) or Alexa Fluor 488 dye; it can also be scaled up for fluorometer-based experiments.

Each FluoReporter Biotin Quantitation Assay Kit for biotinylated proteins includes:

• Biotective Green reagent
• Biocytin
• Protease
• Concentrated phosphate-buffered saline (PBS)
• Biotinylated goat anti–mouse IgG antibody
• Detailed protocols (FluoReporter Biotin Quantitation Assay Kit For Biotinylated Proteins)

Sufficient reagents are provided for assaying 5 samples independently using eight wells in triplicate for the standard curve and three dilutions of the sample in triplicate (totaling 33 wells per assay). However, fewer wells may be used to conserve sample and a single standard curve can be used for multiple samples in the same experimental session. Biocytin (biotinylated lysine) is provided as a standard for the assay because it more closely represents the form of biotin present after proteolytic cleavage. Biotinylated goat anti–mouse IgG antibody is also provided as a positive control and biotinylated protein standard.

FluoReporter Biotin Quantitation Assay Kit for Biotinylated Nucleic Acids

The FluoReporter Biotin Quantitation Assay Kit for biotinylated nucleic acids (F30755) provides a sensitive fluorometric assay for determining the number of biotin labels on a nucleic acid. This assay is based on the displacement of a quencher dye from the biotin binding sites of Biotective Green reagent. The FluoReporter biotin quantitation assay can detect from 4 to 80 picomoles of biotin in a sample, providing a 50-fold higher sensitivity than the HABA biotin binding assay described by Green. Analysis of multiply biotinylated nucleic acids requires a preliminary nuclease digestion step to avoid underestimation caused by steric restriction of avidin binding. This kit can be applied to as little as 13 ng of biotin-labeled nucleic acid (Sensitivity of the FluoReporter Biotin Quantitation Assay Kit for biotinylated nucleic acids with varying degrees of biotinylation—Table 4.3) and is ideal for determining the degree of biotinylation of cDNA samples used in Affymetrix or RLS microarray protocols. The signal window of this assay has a Z' factor of 0.91.

Each FluoReporter Biotin Quantitation Assay Kit for biotinylated nucleic acids includes:

• Biotective Green reagent
• Biotin-dUMP
• Nuclease
• Concentrated phosphate-buffered saline (PBS)
• Biotinylated DNA postive control
• Concentrated nucleic acid digestion buffer
• Detailed protocols (FluoReporter Biotin Quantitation Assay Kit for Biotinylated Nucleic Acids)

Sufficient reagents are provided for assaying 10 samples independently using eight wells in triplicate for the standard curve and three dilutions of the sample in triplicate (totaling 33 wells per assay). However, fewer wells may be used to conserve sample and a single standard curve can be used for multiple samples in the same experimental session.