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.
Refer to notes about the following procedures used by this protocol:
You can also use the short, less-detailed protocol for reference.
I. Preparation of antibody
II. Covalent conjugation
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
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.
Note: it is critical that sodium azide be completely removed from any
antibody: it will react with the Cy5 and prevent conjugation.
Dialyze or exchange over a column the antibody in "Reaction Buffer". Note that the BioRad protein reagent kit reacts spontaneously in "Reaction Buffer"; it is difficult (but not impossible) to determine which column fractions contain the protein by this method... use of a spectrophotometer is preferred. See hints on column separations of nonfluorescent proteins.
Measure the antibody concentration after buffer equilibration. (For IgG, 1 mg/ml has an A(280) of 1.4). If the antibody concentration is less than 1 mg/ml, the conjugation will probably be sub-optimal. If necessary, dilute the antibody to a concentration of 4 mg/ml.
Cy5 is covalently coupled to primary amines (lysines) of the
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.
For column separations, we often use one of two types of pre-poured columns:
For 1.25ml to 2.5ml sample volumes: PD-10 (Sephadex G-25M), Pharmacia Biotech, catalog No. 17-0851-01.
For 0.5 to 1.5ml sample volumnes: KwikSep dextran desalting columns, Pierce, catalog No. 43232.
Cy5 - Cy5-bis-OSU, N,N'-biscarboxypentyl-5,5'-disulfonatoindodicarbocyanine
Amersham Life Science, catalog No. PA15000
DMSO - anyhydrous dimethyl sulfoxide
Aldrich, catalog No. 27,685-5.
Note: keep the DMSO absolutely dry at all times. We keep the bottle in a dessicator. Pour out an amount of DMSO sufficient for your need and then pipette that; don't pipetter directly into the bottle.
NaHCO3 - sodium bicarbonate
J. T. Baker, catalog No. 3508-05, mw 84.01
NaCO3 - sodium carbonate
J. T. Baker, catalog No. 3602-01, mw 106
NaCl - Sodium Chloride
Sigma, Catalog No S-3014, mw 58.44
TRIZMA pre-Set crystals 8.0 - Combination of Tris base and TrisHCl
Sigma, catalog No. T4753, average mw 141.8
NaN3 – Sodium Azide
Sigma, catalog No S-2002, mw 65
500 mM carbonate, pH 9.5
To make 1 Liter:
Note: sodium azide cannot be added to this buffer
10 mM Tris, 150 mM NaCl, 0.1% NaN3, pH 8.2
To make 1 Liter:
1.42g TRIZMA 8.0
pH to 8.2
See hints on storing buffers.