Cy5-SH Of NHS ester PEG Is For Targeted Drug Delivery

The entire conjugation can be performed in about a half-day. In addition to the materials listed below, you will need to have a solution of your antibody at a concentration (optimally) of at least 2 mg/ml. The extent of Cy5 conjugation to the antibody may depend on the concentration of antibody in solution; for consistent conjugations, use a consistent concentration. You should be familiar with how to use a desalting column and how to take absorbance spectra.

The reactive Cy5 molecule is unstable. Open a vial, and weigh out the amount you need (typically, 1 or 2 mg is more than enough). Reseal the vial and store under dessicant at 4C. Immediately disoolve the Cy5 in DMSO at a concentration of 10 mg/ml.

When first conjugating an antibody, a range of Cy5 to antibody concentrations should be compared. We recommend molar ratios of 3, 5, and 7 to start with. Compare each conjugate by staining (you should perform a titration of antibody on cells for each reagent to determine the optimal staining concentration). Select the conjugate with the brightest "positive" cells which still has low background on "negative" cells.

Important Product Information

? Cy5-SH is moisture-sensitive. Store the vial of biotin reagent at 4°C with desiccant. To avoid moisture condensation onto the product, equilibrate vial to room temperature before opening.

? As directed in the procedure, dissolve the NHS ester reagent immediately before use. The NHS moiety readily hydrolyzes and becomes non-reactive; therefore, weigh and dissolve only a small amount of the reagent at a time, and do not prepare stock solutions for storage. Discard any unused reconstituted reagent.

? Avoid buffers containing primary amines (e.g., Tris or glycine) as these will compete with the reaction. If necessary, dialyze or desalt to exchange the protein sample into an amine-free buffer such as phosphate-buffered saline ( PBS ) .

? When Pegylating proteins in solution, excess non-reacted PEG linker is easily removed by size exclusion using either desalting columns or dialysis (Additional Information). A 10mL desalting column is best suited for processing Pegylation reactions involving 1-10mg of protein in approximately 0.5-2mL. For smaller amounts of protein and/or smaller reaction volumes, both the Pegylation reaction and subsequent buffer exchange may be performed in a single Thermo Scientific Slide-A-Lyzer MINI Dialysis Unit. For larger reaction volumes than can be processed with a desalting column, either split the sample between two columns.