AAT Bioquest

How To Determine The Degree of Labeling

The Degree of Labeling (DOL), also referred to as the Degree of Substitution (DOS), plays a crucial role in characterizing and optimizing bioconjugates, particularly those involving fluorophore-labeled proteins. Expressed as a molar ratio representing the label-to-protein proportion, the DOL serves as a valuable parameter for understanding the fluorescence properties of these conjugates. Generally, bioconjugates with lower DOL values exhibit weaker fluorescence intensities, while excessively high DOL values (e.g., DOL > 6) can lead to diminished fluorescence due to self-quenching caused by the fluorophore. Hence, accurately determining the DOL is pivotal for researchers in identifying the optimal bioconjugation ratio for a specific labeling and ensuring consistent and reliable conjugation outcomes.

In the context of antibodies, the ideal DOL range typically falls between 2 and 10. However, the precise value depends on the unique characteristics of the label and protein involved. Therefore, determining the optimal DOL necessitates careful experimental exploration, often involving multiple small-batch labelings. Through such systematic investigations, researchers can pinpoint the most suitable DOL for a given bioconjugation, ensuring both optimal performance and reproducibility in the conjugation process. These endeavors enable researchers to unlock the full potential of bioconjugates, facilitating comprehensive characterizations and maximizing their utility across diverse applications.

For assistance with DOL calculations, use AAT Bioquest's Degree of Labeling (DOL) Calculator.

Key Factors to Consider

  • Prior to absorbance measurements, it is essential to eliminate any residual unbound dye. This can be achieved through thorough dialysis or gel filtration methods.
  • The extinction molar coefficient (ε) of the unlabeled protein must be known.
  • Both the extinction molar coefficient and the wavelength at which the fluorescent dye exhibits maximum absorption also must be known.
  • To ensure accurate determination of protein concentration at 280 nm, it is important to account for the absorption of dyes at the same wavelength. A correction factor can be calculated by dividing the A280 of the dye by its Amax value. This correction factor helps adjust for the contribution of dye absorption at 280 nm.

Protocol to Determine Degree of Protein Labeling

  1. To ensure optimal results and precise determination of the dye:protein ratio, completely remove any unbound dye from the sample by dialysis or gel filtration.
  2. Measure the absorbance of the protein:dye conjugate at 280 nm using a spectrophotometer with a 1 cm path length cuvette.

    Note: If the absorbance measurement is > 2.0, then dilute the sample to achieve values <2.0. Record the dilution factor, this will be required in the calculations.

  3. Measure the absorbance of the protein:dye conjugate at the λmax of the dye (see Table 1).
  4. Calculate the molarity of the protein:
    • ε = protein molar extinction coefficient (e.g., ε of IgG is ~210,000 M-1 cm-1)
    • Amax = absorbance of dye solution measured at the wavelength max of dye
    • CF = correction factor
    • Dilution Factor = amount, if any, to which the protein:dye sample was diluted for absorbance measurement

    Protein concentration (M)   =   A280 - (Amax x CF)   x   dilution factor
  5. Calculate the degree of protein labeling:
    • ε' = molar extinction coefficient of the fluorescent dye

    Moles dye per mole protein   =   Amax of labeled protein   x   dilution factor
    ε' x protein concentration (M)

Table 1. Correction factors and critical values for iFluor®, Alexa Fluor®, and other commonly used dyes.

Molecular Weight
Ex (nm)
Em (nm)
iFluor® 350749.8534545020,0000.230.83
iFluor® 405755.5840342737,0000.770.48
iFluor® 430688.7943349840,0000.30.68
iFluor® 440692.8343448040,0000.2290.352
iFluor® 450603.6945150240,0000.270.45
iFluor® 460793.9946849380,0000.460.98
iFluor® 488945.0749151675,0000.110.21
iFluor® 5141013.9551152775,0000.1160.265
iFluor® 532914.0653756090,0000.160.26
iFluor® 5461145.41541557100,0000.150.25
iFluor® 5551125.26557570100,0000.140.23
iFluor® 5601257.45560571120,0000.0690.0482
iFluor® 5681173.46568587100,0000.150.34
iFluor® 570881.07560571120,0000.0690.048
iFluor® 5941160.42588604180,0000.040.05
iFluor® 5971058.29598618100,0000.5140.335
iFluor® 6101253.52610628110,0000.490.32
iFluor® 6331249.58640654250,0000.0440.062
iFluor® 6471274.66656670250,0000.030.03
iFluor® 6601281.66663678250,0000.080.07
iFluor® 670923.15671682200,0000.0330.03
iFluor® 680957.17684701220,0000.0940.097
iFluor® 6901462.75685704220,0000.060.09
iFluor® 700977.16690713220,0000.040.09
iFluor® 7101206.39717739190,0000.070.12
iFluor® 7201089.29716740240,0000.130.15
iFluor® 7401551.19742764225,0000.160.16
iFluor® 7501416.83757779275,0000.0390.044
iFluor® 7701082.27777797250,0000.080.09
iFluor® 7801526.90784808250,0000.120.13
iFluor® 7901768.30787812250,0000.090.1
iFluor® 8001541.91801820250,0000.080.03
iFluor® 8101576.03811822250,0000.150.09
iFluor® 8201585.92822850250,0000.160.11
iFluor® 8301527.88830867---
iFluor® 8401422836879200,0000.090.2
iFluor® 8601571.90853878250,0000.140.1
iFluor® A71288.58762782275,0000.030.03
XFD350 *Same Structure to Alexa Fluor® 350*410.3534343919,0000.190.25
XFD488 *Same Structure to Alexa Fluor® 488*643.4050052071,0000.110.30
XFD514 *Same Structure to Alexa Fluor® 514*916.0751854380,0000.180.31
XFD532 *Same Structure to Alexa Fluor® 532*723.7753455381,0000.090.24
XFD546 *Same Structure to Alexa Fluor® 546*1159.60561572112,0000.120.21
XFD555 *Same Structure to Alexa Fluor® 555*∼1250553568150,0000.080.08
XFD568 *Same Structure to Alexa Fluor® 568*791.8057960391,3000.460.45
XFD594 *Same Structure to Alexa Fluor® 594*819.8559161890,0000.560.43
XFD647 *Same Structure to Alexa Fluor® 647*1259.66650671239,0000.030.00
XFD680 *Same Structure to Alexa Fluor® 680*∼1200681704184,0000.050.00
XFD700 *Same Structure to Alexa Fluor® 700*∼1400696719192,0000.070.00
XFD750 *Same Structure to Alexa Fluor® 750*∼1300752776240,0000.040.00
DyLight™ 350∼87435343215,0000.144-
DyLight™ 405∼79340042030,0000.564-
DyLight™ 488∼101149351870,0000.147-
DyLight™ 550∼1040562576150,0000.081-
DyLight™ 594∼107859361880,0000.585-
DyLight™ 633∼1066638658170,0000.110-
DyLight™ 650∼1066652672250,0000.037-
DyLight™ 680∼950682715140,0000.128-
DyLight™ 755∼1092755776220,0000.030-
DyLight™ 800∼1050770794270,0000.045-

Original created on May 16, 2023, last updated on May 16, 2023
Tagged under: Antibody conjugation, Protein conjugation, Correction factor, Extinction Coefficient