AFDye 647 NHS ester cell staining

aAFDye 647 NHS ester

(Alexa Fluor 647 NHS ester Equivalent)

Product Information

Product description

Dye 647 is a water-soluble, strong light stable dye with a very high photon yield. AFDye 647 is a bright far-infrared fluorescent dye that can be excited using 633nm or 647nm laser spectral lines. Suitable for imaging low abundance targets, especially in fixed cells. Strong tolerance within the pH range of 4-10.

AFDye647 NHS ester (Equivalent to Alexa Fluor 647 NHS ester) is an amine reactive, far-infrared fluorescent dye that typically uses primary amines to label proteins or antibodies, peptides, amino modified oligonucleotides, and other amino containing biomolecules. This label is usually most effective in the pH range of 7-9, forming stable and covalent amide bonds.

Product Features

Preservation and transportation methods

Storage: Store in the dark and dry at -20 º C for 2 years.

Transportation: Ice pack transportation.

Precautions

1) Fluorescent dyes all have quenching issues, please try to avoid light as much as possible to slow down fluorescence quenching.

2) Although AFDye NHS ester series dyes have a certain degree of water solubility, it is strongly recommended to dissolve them in DMSO and DMF to prepare the mother liquor before conducting subsequent labeling experiments. Because in water-soluble systems, these reactive groups are easily hydrolyzed to form non reactive free acid forms.

3) For your safety and health, please wear lab coats and disposable gloves when operating.

1、 Staining steps (taking the labeling of immunoglobulin IgG as an example)

Almost all proteins or peptides can be labeled with amine reaction reagents (probes). The following are the optimized labeling steps for IgG antibodies. Users can enlarge or shrink the reaction system according to their own protein content, but in order to obtain ideal staining results, the protein concentration must not be less than 2mg/ml. Due to the significant differences in the number of amino groups and surface positions of amino groups in different proteins (peptides), it is recommended that users establish three different amine reaction reagents: protein molar ratios, during the initial experiment to screen for the molar ratio that can achieve the best labeling efficiency.

Dissolve approximately 10mg of protein in 1ml of 0.1M sodium bicarbonate buffer.

Note 1: The protein concentration is usually 5-20mg/ml, and the success rate of conjugation is highly dependent on the protein concentration. When the concentration is below 2 mg/ml, it will seriously affect the labeling efficiency.

Note 2: The labeling system should avoid any amine based substances such as Tris, glycine, ammonium salts, or other stable proteins such as BSA. If the protein solution to be labeled contains contamination from the above substances, it needs to be dialyzed into 10-20 mM phosphate buffered saline (PBS), and then 0.1 ml of 1M bicarbonate buffer (pH 8.3-9.0) should be added to each ml of protein solution to adjust to the desired pH.

Note 3: Low concentrations of sodium azide (<3 mM) or mercury sulfide (<1 mM) present in the antibody solution will not interfere with the labeling reaction.

1.2 Dissolve the amine reaction reagent (probe) (such as AFDye647 NHS ester) in high-quality anhydrous DMF or DMSO to 10 mg/ml.

Note 1: The aqueous solution of AFDye 488 NHS ester must be prepared and used immediately, and cannot be stored, as it will be hydrolyzed into inactive free acids in aqueous solution. The DMSO storage solution of AFDye 488 NHS ester can be stored at ≤ -20 ℃ and is recommended to be used up within 2 weeks.

1.3 Slowly add 50-100 µ l of amine reagent (probe) while stirring or vortexing the protein solution.

Note 1: The amount of amine reagent (probe) added is 0.5-1 mg. Overall, about 1/4-1/3 of the amine reagent will couple to the protein. The labeling effect of different proteins and labeling reagents varies. Please optimize the ratio of the two according to the specific situation.

1.4 Slowly stir at room temperature and incubate in the dark for 1 hour (or longer).

1.5 [Optional] Terminate the reaction by adding 0.1ml of freshly prepared 1.5M hydroxylamine (pH 8.5) and incubate the hydroxylamine containing system at room temperature for 1 hour.

1.6 Purification: Choose a suitable method (such as dialysis, P-30 column, or PD-10 column) to purify the labeled product, removing salt ions or unreacted amine reaction reagents.

1.7 Storage: The labeled protein should be stored in the dark at 2-8 ℃. If the concentration of purified protein conjugate is less than 1mg/ml, add BSA or other stable proteins 1-10mg/ml. In the presence of 2mM sodium azide, it can be stored for several months at 2-8 ℃. If it needs to be stored for a longer time, it should be packaged and stored in the dark at -20 ℃ to avoid repeated freezing and thawing.

2、 Calculate the marking ratio (F/P value)

For most antibodies such as immunoglobulin IgG, a molar absorptivity of 203000 and an F/P value between 4 and 9 are the most suitable.
2.1 AFDye647 labeled protein F/P value calculation:
1) Dilute a small amount of purified conjugated protein with precise multiples of PBS and measure the light absorption at 280nm and 650nm in a 1cm colorimetric dish;
2) Calculate the protein concentration in the sample: protein molar concentration=(A280- (A650 × 0.03)) × dilution factor/203000
3) Calculate the labeling ratio: the number of moles of dye bound per mole of protein=A650 x dilution factor/(239000 x protein molar concentration)

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——Written/Edited by V. Shallan [Copyright belongs to MKBio Maokang]

Shanghai Maokang Biotechnology Co., Ltd. is a company engaged in the research of reagents, instruments, laboratory consumables, and experimental services in the fields of life sciences and biotechnology. It mainly engages in cell biology, botany, molecular biology, immunology, biochemistry, and proteomics. In the fields of biopharmaceuticals and diagnostic reagent research and production. Our company adheres to the business philosophy of "people-oriented, honesty and trustworthiness, and contract keeping". Adhere to the principle of 'quality assurance' to provide high-quality products to our customers.