Fluorescent dye antibody labeling kits are designed for efficiency and reliability, delivering ready-to-use labeled proteins in just 60 minutes, with minimal hands-on time. The kits include all necessary reagents for antibody labeling, including amine-reactive dye, buffer, and purification columns. The reactive dyes contained in these kits readily react with lysine residues in antibodies or proteins to yield a covalently attached fluorescence-based sensor. Spin columns included in the kits are used for purifying the labeled antibody from excess dye with yields of 70–95%. The kits contain sufficient reagents for five labeling reactions of 100 μg of antibody. The wide variety of colors are compatible with various detection systems, including fluorescence microscopy and flow cytometry.
Contents and storage
| A1491935 | Component | 5 reactions | Storage | Quantity Per Reaction |
| A1491935A | AF532 NHS Ester | 5 vials | -20℃. Store in the dark. | 1 vial for labeling 100 μg of antibody |
| A1491935B | NaHCO3 | 100 mg | RT. | Prepare according to instructions
|
| A1491935C | Spin Desalting Column | 5 EA | 2-8℃. Do not freeze. | 1 EA for 1 reaction |
| A1491935D | Collection Tubes | 10 EA | RT. | 2 EA for 1 reaction |
Required materials not supplied
1. Microcentrifuge capable of 1,000 xg.
2. Desired antibody for labeling (free of BSA or any carrier protein).
3. PBS buffer (pH 7.2-7.4).
Important
1. The purified antibody should be in a buffer that does not contain primary amines (for example, ammonium ions, Tris, glycine, ethanolamine, triethylamine, glutathione) or imidazole. All of these substances significantly inhibit protein labeling.
2. Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well.
3. Concentrate the antibody to ≥1 mg/mL.
4. Do not reuse the purification resin.
5. Before opening the vials, warm all components to room temperature.
6. The reactive dyes should be protected from prolonged exposure to light.
Instructions for Use
1. Prepare the reagents
a) Prepare a 1 M sodium bicarbonate solution—Prepare an appropriate amount of 1M NaHCO₃ solution based on the sample volume. For example, weigh 42mg of NaHCO₃ and add 0.5mL of ultrapure water to obtain a 1M NaHCO₃ solution. NaHCO₃ can maintain the pH of the labeling reaction system between 7-9, thereby improving labeling efficiency. Note: The NaHCO₃ solution must be prepared fresh before each use.
2. Label the antibody
a) If the antibody to be labeled has a concentration of ≥1 mg/mL and is in an appropriate buffer, dilute it to 1 mg/mL, and add a 10% volume of 1 M sodium bicarbonate buffer. If the antibody is a powder lyophilized from an appropriate buffer, prepare a 1 mg/mL solution of the antibody by adding an appropriate amount of 0.1 M sodium bicarbonate buffer to the antibody. Prepare 0.1 M sodium bicarbonate buffer by diluting the 1 M solution 10-fold with deionized water.
b) Transfer 100 μL of the antibody solution to the vial of reactive dye. Cap the vial and gently invert it a few times to fully dissolve the dye.
Note: To visually ensure that the dye has fully dissolved, peel the label off the vial of reactive dye.
c) Incubate the solution for 60 minutes at room temperature in the dark. The reaction can be carried out on a shaker or mixer, recommended speed for flipping up and down is 25rpm. If a mixing instrument is not used during the reaction process, the reaction solution should be mixed upside down every 10 minutes.
Note: During the incubation period, proceed to steps 3 below, to prepare a spin column for the purification of the labeled protein.
3. Prepare the spin column
a) Loosen the cap on a spin column, twist the tab off of the bottom, then place the column into a collection tube.
b) Centrifuge the column‑tube assembly at 1,000 x g for 2 minutes to remove the storage solution.
Note: When using a fixed-angle rotor, place a mark on the side of the column that faces away from the rotor center. For all subsequent centrifugation steps, place the column in the microcentrifuge with the mark facing away from the rotor center.
c) Discard the flowthrough, then place the column back into the collection tube.
d) Add 500 μL of PBS Buffer, then centrifuge the column‑tube assembly at 1,000 x g for 2 minutes to equilibrate the column. Repeat this step d) 3 times, discarding the buffer from the collection tube each time.
4. Purification with Desalting Column
a) Transfer the equilibrated column into a new collection tube.
b) Carefully pipette the entire reaction mixture onto the center of the column.
c) Centrifuge the column‑tube assembly at 1,000 × g for 2 minutes. The purified antibody conjugate is in the collection tube.
5. Determine the antibody concentration and DOL (Optional)
The degree of labeling (DOL) is a useful parameter for characterizing the average number of label molecules that have covalently bonded to your sample protein during the labeling reaction. It can be determined from the absorption spectrum of your labeled bioconjugate.
a) Detect the absorbance of the antibody conjugate at 280 nm (A ₂₈₀ ) and the absorbance maximum (λmax) for the respective dye (Adye). The λmax values for commonly used fluorophores are given in the table below.
| Cat.No. | Dyes | λmax (nm) | Em (nm) | εdye (M⁻¹cm⁻¹) | CF ₂₈₀ | Target DOL | MW (g/mol) |
| A1491932 | AF350 | 343 | 441 | 19,000 | 0.19 | 4-9 | 410 |
| A1491933 | AF405 | 401 | 422 | 34,000 | 0.7 | 4-9 | 1028 |
| A1491934 | AF488 | 490 | 525 | 76,000 | 0.1 | 4-9 | 1081 |
| A1491935 | AF532 | 530 | 555 | 81,000 | 0.09 | 4-9 | 723 |
| A1491936 | AF555 | 553 | 568 | 150,000 | 0.08 | 4-8 | 1250 |
| A1491937 | AF594 | 590 | 618 | 92,000 | 0.56 | 4-9 | 820 |
| A1491938 | AF647 | 650 | 668 | 239,000 | 0.03 | 4-8 | 1300 |
| A1491939 | AF660 | 663 | 691 | 132,000 | 0.1 | 3-8 | 1100 |
| A1491940 | AF680 | 681 | 704 | 184,000 | 0.05 | 3-7 | 1150 |
| A1491941 | AF700 | 696 | 719 | 192,000 | 0.07 | 3-7 | 1400 |
| A1491942 | AF750 | 752 | 776 | 240,000 | 0.04 | 3-8 | 1300 |
| F1491944 | FITC | 498 | 517 | 73,000 | 0.35 | 3-8 | 389 |
| P1491945 | Pacific Blue | 410 | 455 | 46,000 | 0.2 | 4-9 | 339 |
| O1491947 | Oregon Green 488 | 498 | 526 | 76,000 | 0.12 | 3-8 | 509 |
| T1491948 | TRITC | 557 | 576 | 100,000 | 0.34 | 1-3 | 443 |
| C1491949 | Cy3 | 554 | 566 | 150,000 | 0.07 | 3-5 | 734 |
| C1491950 | Cy5 | 647 | 665 | 250,000 | 0.03 | 3-5 | 754 |
Note:
1. Target DOL is the acceptable degrees of labeling (DOL) for a whole IgG.
b) The following formula can be used to calculate the antibody concentration:
Antibody concentration (mg/mL) = (A280 - CF280 × Adye) / 1.4
c) The following formula can be used to calculate the degree of labeling:
DOL = (Adye / εdye) / [(A280 - CF280 × A280) / 210,000]
Note:
1. 210,000 is the molar extinction coefficient (Ec) in M-1cm-1 of IgG at 280nm.
2. Adye is the absorbance at maximum (λmax) for the respective dye.
3. CF280 is the correction factor for the effect of the fluorophore on absorbance at 280nm.
6. Strorage
a) Add 0.05–0.2% Proclin 300 or 0.05% sodium azide, along with a protein stabilizer (such as 0.1% BSA), to the labeled protein. Store protected from light at 2–8°C for stable preservation up to six months. Alternatively, add an equal volume of glycerol and store at -20°C for stable preservation up to six months.
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