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Protocol: Maleimide Labeling of Protein Thiols

Dyes functionalized with a maleimide group can react with thiol groups to form thioether-coupled products. The reaction can take place at neutral pH, and is not affected by the presence of amines. At neutral pH, the maleimide group does not react with histidine or arginine. Maleimide labeling of antibodies can be used as an alternative to succinimidyl ester labeling of amines, for antibodies where amine labeling affects the antibody binding affinity. Also see our protocols for Succinimidyl Ester Labeling of Protein Amines and Aminooxy Labeling of Glycoproteins.

The protocol below is a typical procedure for labeling IgG antibodies. The protocol can be adapted to us other dye or biotin maleimide. Protocols for labeling other thiol- containing molecules are similar, except for the purification procedures, which may need to be modified for different molecules. The labeling reaction may be scaled up or down for any amount of protein as long as the ratios of the reagents are maintained.

Materials required:

Workflow overview:

  1. Prepare the antibody for labeling
  2. Optional: Reduce disulfide bonds with TCEP (~30 minutes)
  3. Prepare the dye stock solution
  4. Perform the labeling reaction (2 hours to overnight)
  5. Purify the conjugated antibody
  6. Calculate the degree of labeling
  7. Add antibody stabilizers to conjugate


1 – Prepare antibody solution for labeling

Dissolve the antibody at 50-100 uM (7.5-15 mg/mL for IgG) in 1X PBS or 10-100 mM phosphate, Tris, or HEPES buffer, pH 7-7.5. As an optional step, if you wish to free up more thiol groups from the disulfide bonds in the protein, you may add ~10-fold molar excess of TCEP at this stage. Incubate the reaction solution for ~30 minutes. The reduction reaction and the subsequent labeling reaction are best carried out in the presence of an inert gas (N2 or Ar) to prevent re-formation of disulfide bonds.

2 – Prepare dye stock solution

Allow the vial of CF® dye maleimide to warm up to room temperature. Prepare a 10 mM dye stock solution. For 1 umol dye: add 100 uL anhydrous DMSO to the vial. For 0.25 umol dye: add 25 uL anhydrous DMSO to the vial. Vortex the vial briefly to fully dissolve the dye, followed by brief centrifugation to collect the dye at the bottom of the vial.

  • If the labeling reaction is to be carried out with a small amount of protein, the dye stock solution may need to be more dilute for accurate pipetting.
  • Unused stock solution may be stored at -20°C, protected from light and moisture. If anhydrous DMSO is used for making the solution, the dye should be stable for at least one month.
  • Dye stock solution may also be prepared in dH2O or aqueous buffer. However, because the dye will hydrolyze over time, aqueous stock solutions should be prepared immediately before the conjugation reaction and cannot be stored for later use.

3 – Carry out the labeling reaction

While stirring or vortexing the protein solution, add a volume of dye stock solution to result in a dye:protein molar ratio of 10-20 dyes per protein. For example, for IgG at 50 uM, you would add 50-100 uL of 10 mM dye for every 1 mL of antibody solution, for a final dye concentration of 0.5-1 mM. Continue to stir or rock the reaction solution at room temperature for 2 hours or at 4°C overnight, protected from light.

  • While the labeling reaction is underway, proceed to step 4 to prepare a Sephadex® column. See Table 1 for the appropriate Sephadex® medium to use for each CF® dye.

4 – Separate the labeled protein from the free dye

Prepare a Sephadex® column (10 mm x 300 mm) equilibrated in 1X PBS buffer. Load the reaction solution from step 3 onto the column and elute with PBS buffer. The first band excluded from the column corresponds to the antibody conjugate.

  • For small scale labeling reactions, you may use an ultrafiltration vial to remove the free dye from the conjugate in order to avoid an overly dilute product. 10K MWCO vials can be used for IgG; proteins with different molecular weights may require different MWCO.

5 – Determination of degree of labeling (DOL)

Degree of labeling (DOL) is the average number of dye molecules conjugated to each antibody molecule. Measure the absorbance of the eluted antibody conjugate solution at 280 nm and at the absorption maximum of the dye used for labeling. The optimal range of DOL for each dye is listed in Table 1, although a DOL slightly above or below this range will also produce good results.

  • The protein solution eluted from the column may be too concentrated for accurate absorbance measurement and thus must be diluted to approximately ~0.1 mg/mL. The fold of dilution (“dilution factor”) necessary can be estimated from the amount of starting antibody (i.e., 5 mg) and the total volume of the protein solution collected from the column.

5a. Determine the protein concentration

The concentration of the antibody conjugate can be calculated from the formula:
[conjugate] = {[A280 – (Amax x Cf)]/1.4} x dilution factor

where [conjugate] is the concentration in mg/mL of the antibody conjugate collected from the column; “dilution factor” is the fold of dilution used for spectral measurement; A280 and Amax are the absorbance readings of the conjugate at 280 nm and the absorption maximum respectively; Cf is the absorbance correction factor; and the value 1.4 is the extinction coefficient of IgG in mL/mg. See Table 1 for the Amax and correction factor for each CF® dye.

  • If labeling a protein other than IgG, use the extinction coefficient for that specific protein.
  • If using a dye other than a CF® dye, use the Amax and correction factor for that specific dye.

5b. Calculate the degree of labeling (DOL):

The DOL is calculated according to the formula:
DOL = (Amax x Mwt x dilution factor)/(ε x [conjugate])

where Amax, “dilution factor” and [conjugate] are as defined in Step 5a, Mwt is the molecular weight of IgG (~150,000), and ε is the molar extinction coefficient of the dye (see Table 1).

  • If labeling a protein other than IgG, use the molecular weight for that specific protein.
  • If using a dye other than a CF® dye, use the extinction coefficient for that specific dye.

6 – Storage and handling of labeled antibody

For long-term storage, we recommend adding 5-10 mg/mL BSA and 0.01-0.03% sodium azide to the conjugate solution to prevent denaturation and microbial growth. The conjugate should be stored at 4°C, protected from light. If glycerol is added to a final concentration of 50%, the conjugate can be stored at -20°C. Under these conditions, antibody conjugates are stable for a year or longer.

Table 1: CF® Dye Technical Data

DyeAbs/Em (nm)Sephadex® mediaAmax (nm)Cf (protein)εOptimal DOL (IgG)
CF®555555/565G-255550.08150,0004-5, 3-6 ok
CF®647650/665G-256500.03240,0004-5, 3-6 ok
CF®660C667/685G-756670.08200,0003-6, 2-3 ok
CF®680681/698G-756810.09210,0003-5, 2-3 ok
CF®750755/777G-757550.03250,0003-5, 2-3 ok
CF®770770/797G-757700.06220,0003-5, 2-3 ok
Sephadex is a registered trademark of GE Healthcare.