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DiSulfo – Cy5 hydrazide is a far-red dye with hydrazide group. The dye can be used as a polar tracer or for labeling biomolecule with an aldehyde or ketone group (such as carbohydrate molecules after peroxidation with periodate). Examples are proteins subjected to oxidative stress, and oligonucleotides with aldehyde moieties. DiSulfo – Cy5 hydrazide replaces carbonyl-reactive Alexa Fluor 647, DyLight 650 and CF ™ 647 hydrazide dyes.
Cy ™ - GE Healthcare Companу (previously Amersham Biosciences Corp.)DyLight ® - Thermo Fisher Scientific Inc.AlexaFluor ® - Molecular Probes Inc. (Life Technologies Corp.)CF ™ - Biotium Inc.
Packs, each containing dried dye to label 1 mg of antibody. Conjugation buffer: phosphate buffer pH 7.0
1. All solvents and buffers should be degassed This protocol has been designed for the preparation of diSulfo - Cy5-labelled IgG antibodies. It is designed to label 1 mg. This assumes an average protein molecular weight of 155 K Daltons.
1. Dissolve the antibody in coupling buffer (10 mg/ml). Place 1 mg of antibody to be labelled (0.1 ml) in a 0.5 ml microfuge tube and ﬂush with nitrogen.
2. Add 150 µl Sodium Periodate solution (0.1 M) to the antibody, seal tightly and mix thorough for 30–45 minutes in the absence of light at ambient temperature.
3. While the oxidation reaction is incubating, equilibrate the gel ﬁltration column in coupling buffer. Place the periodate reaction on the column: collect and combine the fractions containing antibody
1. Dissolve the diSulfo - Cy5 Hydrazide in coupling buffer to afford a 0.02 M solution.
2. Combine equal volumes of antibody to diSulfo - Cy5 Hydrazide solution and gently mix for 3 hours at ambient temperature or overnight at 4°C.
3. Purify the product using a 10 ml gel ﬁltration column that has been pre-treated with PBS. Elute the product with PBS.
4. Isolate the ﬁrst coloured fractions and scan sample from 250–800 nm against a PBS blank.
1. Unconjugated dye can also be separated from the labelled antibody by dialysis. Dialysis does not give as efﬁcient and rapid separation as gel ﬁltration. It is recommended that gel ﬁltration be used whenever possible.
Materials supplied Dried dye to label 1 mg of carbonyl compound.
Materials required but not supplied Conjugation buffer: 1:1 Methanol/Triethylammonium acetate buffer, pH 8.0 Sigma-Aldrich (90358).
1. The pH will change the labelling efficiency of the reaction. Optimal labelling occurs at pH 7.2.
2. All solvents and buffers should be degassed. This protocol was used to label ~7.8 µmol of octyl aldehyde with Cy5 hydrazide reagent.
1. Make a solution of diSulfo - Cy5 hydrazide in a 1:1 Methanol/TEAA buffer, pH 8.0 solution to 1 mg/ml and flush with nitrogen.
2. Make a solution of the carbonyl derivative in a 1:1 Methanol/TEAA buffer solution pH 8.0 to 1 mg/ml and flush with nitrogen.
3. Add together the 2 solutions and gently mix for 3 hours.
4. Remove the reaction solvent in vacuo.
1. Purify the product by reverse phase HPLC. Monitor at the wavelength of diSulfo - Cy5, 650 nm.
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