ab188290 GOLD-Oligonucleotide Conjugation Kit Instructions for Use For the Covalent Conjugation of Oligonucleotides to Gold This product is for research use only and is not intended for diagnostic use. Version 2 Last Updated 8 May 2014
Table of Contents 1. Introduction 2 2. Kit Contents 3 3. Storage and Handling 4 4. Additional Materials 4 5. General Guidelines 5 6. Labeling Protocol 6 7. Frequently Asked Questions 8 1
1. Introduction Oligonucleotides conjugated with GOLD nanoparticles have arisen as next generation materials for biological sensing as well as for bottom-up nanotechnologies based on the base pairing principle. The Abcam GOLD-Oligonucleotide Conjugation Kit (ab188290), in combination with a bio-polymer GOLD coating technology, allows sulfhydryl-oligonucleotides to be covalently attached to ultra-stable* GOLD nanoparticles at very high OD quickly and easily. The handson time for the conjugation procedure is about 5 minutes and the conjugate is ready to use within 2 hours. *The bio-polymer surface coat does not detach from gold surfaces even under the most extreme conditions. The conjugation reaction is initiated simply by adding a solution of the sulfhydryl-activated oligonucleotide to the GOLD nanoparticles. The oligonucleotides must be between 10 and 120 base pairs in length and contain a terminal sulfhydryl group, which must be added during synthesis. All commercial oligo suppliers offer this modification. Free thiols must be excluded from samples and reaction buffers, because they will compete for coupling sites. The resulting covalent conjugates are far more stable than those prepared by adsorption of thiolated oligonucleotides to bare metal surfaces. 2
2. Kit Contents Components Amount Storage 1 Test 3 Tests Gold Particles for Oligo Conjugation 1 glass vial 3 glass vials -20 C Gold/Oligo Reaction Buffer 1 vial 1 vial -20 C Gold/Oligo Antibody Diluent 1 vial 1 vial -20 C Gold/Oligo Quencher Reagent 1 vial 1 vial -20 C 3
3. Storage and Handling The kit should be stored at -20 C upon receipt and once opened, stored the unused vial with silica gel (the vials are moisture sensitive). For handling refer to Safety Datasheet 4. Additional Materials Pipettes and pipette tips Microfuge Tubes (0.5 or 1.5 ml) Microfuge 4
5. General Guidelines A. Prior to Labeling Oligonucleotide must be HPLC purified and contain a terminal sulfhydryl group. Avoid thiols (e.g. mercaptoethanol, DTT) Note: The kit is compatible with PBS, MES, MOPS, HEPES, sugars, salts and detergents. B. Amount of Oligonucleotide The optimum amount of oligonucleotide (which will influence the number of oligos per particle) may be application-dependent and must be determined by experimentation: you may need to conjugate different amounts of oligo to optimize your assay. Stock oligonucleotide at concentrations of >100uM are recommended; as this allows potentially interfering substances in the oligo preparation to be diluted out (see Section 6; labeling protocol). 5
6. Labeling Protocol 1. Allow all of the reagents to warm to room temperature. 2. Dilute the oligonucleotide in the Gold/Oligo Antibody Diluent into a final volume of 40 µl (3 reaction kit) or 400 µl (1 reaction kit). Add 5 µl or 50 µl of Gold/Oligo Reaction Buffer respectively. Mix gently. 3. Remove the cap from the vial of Gold Particles for Oligo Conjugation, mix and pipette the sample (with added the Reaction Buffer) directly onto the lyophilized material. Resuspend gently by withdrawing and re-dispensing the liquid once or twice using a pipette. 4. Place the cap back on the vial and leave it standing for 1 hour at room temperature (20-25 C); longer incubation time has no negative effect on the conjugate. 5. After incubating for 1 hour (or more), add 5µl (3 reaction kit) or 50 µl (1 reaction kit) of Gold/Oligo Quencher Reagent (supplied as a lyophilized product, take in solution in 100 µl of water and store surplus Gold/Oligo Quencher Reagent at -20 C after use). The conjugate can be used after 20 minutes. You now have 50 µl (3 reaction kit) and 500 µl (1 reaction kit) of 20 OD conjugate. 6. Dilute further as required for your application or if you wish to exchange the Oligo-GOLD into a specific buffer for your assay or test and remove traces of free oligos, centrifuge the 6
conjugate in a microfuge at 9000g for 6 minutes. Carefully remove the supernatant and add your preferred buffer. It is important to avoid substances that have a very high affinity for gold (e.g. thiols). 7. Oligo-conjugates should be stored at +4 C. A preservative may be desirable for long-term storage. 7
7. Frequently Asked Questions What can I do if my oligonucleotide preparation contains interfering substances? For oligos longer than 20 mer the simplest procedure is to desalt them in to a suitable buffer. Relatively weak buffers (e.g. 20 mm) are preferred so that the ph conditions of the covalent reaction are not significantly altered upon addition of the oligo. Does the oligonucleotide bind to the metal surface? No. The protective surface coat completely shields the metal surface and prevents direct metal-antibody interactions. What type of linkage to gold is formed? The oligonucleotide becomes covalently and irreversibly attached via the terminal sulfhydryl group to the bio-polymer surface coating. What if I need bulk material? The kit that you have purchased is a convenience product for rapid production of small quantities of conjugate for screening purposes. Abcam offers the kit in a range of sizes. Please enquire. 8
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