Cy5 With Cas.146368-15-2 Of NHS ester PEG Is Applied In Bioconjugation

Cy5 is a small organic molecule, and is typically conjugated to proteins via primary amines (i.e., lysines). Usually, between 3 and 7 Cy5 molecules are conjugated to each antibody (optimal is usually 5); higher conjugations can result in solubility problems as well as internal quenching (and reduced brightness). Thus, an antibody will usually be conjugated in several parallel reactions to different amounts of Cy5, and the resulting reagents will be compared for brightness (and background stickiness) to choose the optimal conjugation ratio. Cy5 is typically excited by the 633 nm line of HeNe laser, and emission is collected at 680 nm.

Cy5 is covalently coupled to primary amines (lysines) of the immunoglobulin.

Dissolve the Cy5 in anhydrous DMSO immediately before use, at a concentration of 10 mg/ml. Do not delay between weighing out the Cy5 and dissolving it in DMSO; likewise, do not delay the addition of the solubilized material to the antibody.

For the optimal ratio of 5:1, add 40 μg Cy5 per mg of antibody; mix immediately. (See notes above about using different molar rations of Cy5 to antibody).

Wrap the tube in foil; incubate and rotate at room temperature for 1 hour.

Remove the unreacted Cy5 and exchange the antibody into "Storage Buffer" by gel filtration or dialysis.