Protocol: Succinimidyl Ester Labeling of Protein Amines

Dyes functionalized with succinimidyl ester (NHS Ester or SE) groups can be used to covalently label proteins on primary amines (typically lysine residues) to form a stable amide linkage. This is a common method for preparing fluorescently-labeled antibody conjugates. The protocol below is a typical procedure for labeling IgG antibodies in bicarbonate buffer. The protocol can be adapted for using other dyes or biotin SE. Also see our CF® Dye & Biotin SE Protein Labeling Kits, which include everything you need for antibody labeling and purification. Please note that conjugation to amine groups can affect protein function or antibody binding affinity. For alternative antibody labeling chemistries, see our protocols for Maleimide Labeling of Protein Thiols and Aminooxy Labeling of Glycoproteins.

Note: This protocol is not recommended for labeling IgM antibodies. For labeling IgM antibodies, we recommend using our Mix-n-Stain™ CF® Dye IgM Antibody Labeling Kits.

Procedure:

1 – Prepare antibody solution for labeling

Dissolve the antibody in 0.1 M sodium bicarbonate buffer (pH~8.3) at 2.5 mg/mL. If the IgG is already dissolved in a buffer such as PBS, adjust the buffer composition by adding one-tenth volume of 1 M sodium bicarbonate solution (pH 8.3) to the IgG solution for a final bicarbonate concentration of 0.1 M.

• The IgG should be free of any amine-containing stabilizers, such as amino acids, Tris, BSA or gelatin, as these substances will also react with the dye. Small molecules like Tris or amino acids can be removed by dialyzing the antibody against 1X PBS buffer, or using an ultrafiltration vial to exchange the buffer. Dialysis or ultrafiltration will not remove proteins like BSA or gelatin; if present, the antibody must be purified to remove other proteins before labeling.
• Sodium azide does not affect the labeling reaction.
• At about 2.5 mg/mL protein concentration, the labeling efficiency (proportion of the dye that will react with protein) is generally around 35%. A protein concentration of less than 2.5 mg/mL is also suitable for labeling, although the labeling efficiency will be lower. A labeling efficiency of 20-30% can be expected with a protein concentration around 1 mg/mL. Even higher labeling efficiency is possible with protein concentration higher than 5 mg/mL. Because of variations in buffer and protein purity, accurate labeling efficiency can only be determined under your exact conditions. If the IgG solution is too dilute, it may be concentrated using an ultrafiltration vial with 10 kDa molecular weight cut-off (10K MWCO; see related products).

2 – Prepare dye stock solution

Allow the vial of CF® dye SE 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.

• 1 umol dye is sufficient to label 8-15 mg IgG; 0.25 umol dye is sufficient to label 2-3 mg IgG.
• The dye/protein ratio may need to be higher for a more dilute protein solution because of the lower labeling efficiency for more dilute reactants. See Table 1 for the optimal degree of labeling or DOL (number of dyes conjugated to each protein) for each CF® dye. A DOL slightly above or below the optimal range will also produce good results.
• 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

Add 15-25 uL of 10 mM dye stock for every 1 mL of antibody solution. While stirring or vortexing the protein solution, add the dye stock solution in a dropwise fashion. Continue to stir or rock the reaction solution at room temperature for 1 hour, protected from light.

• These volumes correspond to dye/protein molar ratios between 9:1 to 15:1.
• While the labeling reaction is underway, Prepare a Sephadex® column (10 mm x 300 mm) equilibrated in 1X PBS buffer. See Table 1 for the appropriate Sephadex® medium to use for each CF® dye.

4 – Separate the labeled protein from the free dye

Immediately load the reaction solution onto the column and elute with 1X 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 can be used for IgG; proteins with different molecular weights may require different MWCO.
• If you choose not to separate the labeled antibody from the free dye immediately after the reaction, you may add 50 uL of 1 M lysine to stop the reaction.

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