Fluorescent Peptide Labeling Reagents

Introduction

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Although DABCYL has been used to develop a variety of FRET applications, its low quenching efficiency of longer wavelength dyes (such as fluoresceins, rhodamines and cyanines) has limited its use in the development of sensitive fluorogenic FRET probes. Additionally, the absorption spectrum of DABCYL is environment-sensitive. AAT Bioquest has developed the robust Tide Quencher™ (TQ) acceptor dyes for the development of longer wavelength FRET probes. These Tide Quencher™ dark FRET acceptors (such as TQ1, TQ2, TQ3, TQ4, TQ5, TQ6 and TQ7) are optimized to pair with our Tide Fluor™ dyes and the other fluorophores (such as Alexa Fluor^® dyes, AMCA, EDANS, FAM, TAMRA, HEX, JOE, TET, ROX, Cy3, Cy5 and Cy7). Like our Tide Fluor™ (TF) donor dyes, our Tide Quencher™ acceptor dyes are much more cost-effective with comparable or even better performance for your desired biological applications than other similar products on the market. Besides their broad applications in the development of real time PCR probes, our Tide Quencher™ dyes have also been used to develop various protease substrates such as HIV protease (see above), MMPs and secretases. In some cases, it has demonstrated greatly improved enzyme performance. This may be partly due to the red-shifted absorption spectrum that overlaps better with the emission spectrum of fluoresceins, rhodamines and cyanines. Tide Quencher™ dyes are great choice for you to eliminate the limitations of classic quenchers. They are excellent dark quenchers that are individually optimized to pair with all of the popular fluorescent dyes such as fluoresceins and rhodamines. Our TQ dye series are essentially nonfluorescent, and cover the full visible spectrum with unusually high efficiency. For example, TQ2 has absorption maximum perfectly matching the emission of FAM while TQ3 is proven to be the best quencher for TAMRA and Cy3.

In summary, our Tide Quencher™ dyes have the following advantages:

• Most Powerful: enable you to explore the FRET potentials that might be impossible with other quenchers.
• Versatile Reactive Forms: convenient for self-constructing your desired FRET biomolecules.
• A Complete Set of Dyes: perfectly match your desired fluorescent donors.
• Enhanced Value: competitive price with the best performance.

Labeling Mechanism

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Succinimidyl esters are proven to be the best reagents for amine modifications because the amide bonds that are formed are essentially identical to, and as stable as the natural peptide bonds. These reagents are generally stable and show good reactivity and selectivity with aliphatic amines. There are few factors that need be considered when SE compounds are used for conjugation reaction:

1. Solvents: For the most part, reactive dyes are hydrophobic molecules and should be dissolved in anhydrous dimethylformamide (DMF) or dimethylsulfoxide (DMSO).
2. Reaction pH: The labeling reactions of amines with succinimidyl esters are strongly pH dependent. Amine-reactive reagents react with non-protonated aliphatic amine groups, including the terminal amines of proteins and the −-amino groups of lysines. Thus amine acylation reactions are usually carried out above pH 7.5. Protein modifications by succinimidyl esters can typically be done at pH 7.5-8.5, whereas isothiocyanates may require a pH 9.0-10.0 for optimal conjugations.
3. Reaction Buffers: Buffers that contain free amines such as Tris and glycine and thiol compounds must be avoided when using an amine-reactive reagent. Ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation must also be removed (such as viadinlysis) before performing dye conjugations.
4. Reaction Temperature: Most conjugations are done at room temperature. However, either elevated or reduced temperature may be required for a particular labeling reaction

Storage and Handling

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The dye labeled oligos should be stored at ≤–15°C, and kept from light. For longer storage, dye labeled oligo could be divided as single-used aliquot and stored at ≤–15°C. PROTECT FROM LIGHT.

Sample Protocols (For reference purposes only)

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Always wear the protection apparatus (such as gloves). Warm all the components before opening them, and immediately prepare the required solutions before starting the conjugation. Avoid repeated freezing and thawing if possible. Any solutions containing the dye should be kept from light.

Label Amino-Modified Oligonucleotides with Tide Quencher™ Dyes

The following protocol has been optimized for labeling 200 µg (~6 A260 nm units) of a proprietary oligonucleotide. You need modify the protocol to get the best results for your particular application by multiple experimentations. YOUR AMINO-MODIFIED OLIGO MUST BE TREATED TO REMOVE AMMONIA THAT RAPIDLY REACTS AND CONSUMES DYE SUCCINIMIDYL ESTERS.

1. Prepare Oligo Solution (Solution A)
   1. Dissolve your amino-modified oligo (~200 µg) in a tetraborate buffer (100 µL, pH 8.5±0.5).
      Note 1: The oligonucleotide must be synthesized with an amine group on the 5' end. See Appenxidx 1 for the purification of amino-modified oligos.
      Note 2: Avoid buffers that contain primary amines, such as Tris, as these compete for conjugation with the amine-reactive compound.
2. Prepare Dye Solution (Solution B)
   1. Dissolve 1 mg dye SE in 100 µL DMSO (>10 mg/mL if possible) by pipetting up and down. Centrifuge the solution stock on the sides of the vial to the vial bottom.
      Note: prepare the DMSO dye solution before starting the conjugation. Extended storage of the dye solution may reduce the dye activity. Any solutions containing the dye should be kept from light. We do not recommend that you store the DMSO dye solution for future use.
3. Run Conjugation Reaction
   1. To the dye solution (B, 20-50 µL) add the oligo solution (A, 100 µL) with stirring or shaking (keeping the reaction mixture from light).
   2. Rotate or shake the reaction mixture for 4-6 hours at room temperature on a rotator or shaker.
      Note: Gently vortex tap the vial every 10 minutes for the first hour to ensure that the reaction solution remains well mixed. Do not mix violently, as material may be left on the sides of the vial. After six hours, 50–90% of the amine-modified oligonucleotide molecules should be labeled. The reaction might be incubated overnight if it is more convenient. However, overnight incubation will not result in a greater labeling efficiency in most cases.
4. Purify Dye-Oligo Conjugate
   1. Preliminary purification by ethanol precipitation of labeled oligonucleotide
      1. Add 20 µL (one-tenth reaction solution volume in general) of 3 M NaCl and 300 µL cold absolute ethanol (two and half reaction solution volume volumes in general) to the reaction vial.
      2. Mix the solution well and place it at –20°C for 30 minutes.
      3. Centrifuge the solution in a microcentrifuge at 10,000 to 15,000 × g for 30 minutes.
         Note: Loss of sample may occur if the centrifugation is not long enough.
      4. Carefully remove the supernatant, rinse the pellet 1-3 times with cold 70% ethanol and dry briefly.
         Note: Some unreacted labeling reagent may have precipitated over the course of the reaction or may be stuck on the walls of the reaction vial. This material should be completely redissolved by extensive vortex mixing before centrifugation. Redissolving the labeling reagent ensures that the precipitated oligonucleotide will be minimally contaminated with unreacted label.
   2. Final Purification by HPLC or by gel electrophoresis See Appendix I

Label Peptides with Tide Quencher™ Dyes

The following protocol has been optimized for labeling 10 mg of a proprietary peptide (MW ~ 2000) that contains only a single free amino group. YOU NEED MODIFY THE PROTOCOL TO ARCHIE THE BEST RESULTS FOR YOUR PARTICULAR APPLICATION BY MULTIPLE EXPERIMENTATIONS.

1. Prepare Peptide Solution (Solution A) Dissolve your peptide (~10 mg) in DMF (~1 ml).
   Note 1: The peptide must be neutralized with a base such as triethylamine or potassium carbonate.
   Note 2: Avoid buffers that contain primary amines, such as Tris, as these compete for conjugation with the amine-reactive compound.
2. Prepare Dye Solution (Solution B) Dissolve 5 mg dye SE in 500 µL DMF (>10 mg/mL if possible) by pipetting up and down.
   Note: prepare the DMF dye solution before starting the conjugation. Extended storage of the dye solution may reduce the dye activity. Any solutions containing the dye should be kept from light. We do not recommend that you store the DMF dye solution for future use.
3. Run Conjugation Reaction 3.3 To the dye solution (B, 500 µL) add the peptide solution (A, 1 mL) with stirring or shaking (keeping the reaction mixture from light).
   1. Stir the reaction mixture for 4-6 hours at room temperature.
4. Purify Dye-Peptide Conjugate The reaction solution was concentrated and purified on a C18 column to afford the desired conjugate. The fractions were analyzed by HPLC, and the fractions of >97% purity were pooled and lyophilized.
   Note 1: HPLC Purification Conditions: TEAB buffer (triethyl ammonium bicarbonate, 0.25 mmol, pH=7.0-8.0) was used as buffer A and acetonitrile as buffer B. The HPLC was run from 0% B to 30% B in 60 min (flow rate: 100 mL/min).
   Note 2: Avoid strong light during the operation.

References

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1. Hermanson GT (1996). Biocojugate Techniques, Academic Press, New York.
2. Sambrook J., Fritsch E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory.
3. Brinkley M (1992). A brief survey of methods for preparing protein conjugates with dyes, haptens, and cross-linking reagents. Bioconjug Chem 3, 2-13.