AF350 Antibody Labeling Kit

Cat. No.: A1491932

GRADE & PURITY BioReagent

Overview

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

A1491932	Component	5 reactions	Storage	Quantity Per Reaction
A1491932A	AF350 NHS Ester	5 vials	-20℃.  Store in the dark.	1 vial for labeling 100 μg of antibody
A1491932B	NaHCO3	100 mg	RT.	Prepare according to instructions
A1491932C	Spin Desalting Column	5 EA	2-8℃.  Do not freeze.	1 EA for 1 reaction
A1491932D	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 (A₂₈₀) 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)	CF₂₈₀	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 × A_dye ) / 1.4

c)     The following formula can be used to calculate the degree of labeling:

DOL = (A_dye / ε_dye ) / [(A₂₈₀ - CF₂₈₀ × A₂₈₀ ) / 210,000]

Note:

1.210,000 is the molar extinction coefficient (Ec) in M⁻¹cm⁻¹  of IgG at 280nm.

2. A_dye 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.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light,Room temperature,Store at -20°C

Shipped In

Wet ice

Stability And Storage

Each component has a shelf life of 1 year under corresponding storage conditions. A1491932A: Store at -20℃ long term (12 months). Store in the dark. A1491932B: Store at room temperature long term (12 months). A1491932C: Store at 2-8℃ long term (12 mont

Images

Flow Cytometry: AF350 Antibody Labeling Kit (A1491932)

AF350 Antibody Labeling Kit (A1491932) - Flow Cytometry

Flow Cytometry analysis of Jurkat cells stained with 0.5 μg/mL Recombinant CD45 Antibody (Ab210608). Followed by a goat anti-mouse IgG conjugated to AF350 using the AF350 Antibody Labeling Kit (A1491932) at a dilution of 1/300 for 1 hour at room temperature in the dark. Blue - Isotype control, Mouse IgG (Ab170221). Black - Unlabelled control, cells without incubation with primary antibody.