FITC异硫氰酸荧光素酯,3326-32-7

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FITC

CAS No. : 3326-32-7

MCE 国际站：FITC

产品活性：FITC，英文全称 Fluorescein Isothiocyanate，中文名称异硫氰酸荧光素，是生物学中应用最为广泛的一种绿色荧光素衍生物。FITC 具有高吸收率、优良的荧光量子产率和良好的水溶性等特点。FITC 的异硫氰酸基团可与蛋白质上的氨基、巯基、咪唑、酪氨酰、羰基等基团相结合，从而实现包括抗体，凝集素在内的蛋白标记。FITC 除了用作蛋白质标记物，还可用作蛋白质荧光示踪剂，通过标记抗体而快速鉴定病原体，或是用于蛋白质和多肽（HPLC）的微量测序。FITC 的最大激发波长为 494 nm。一旦激发，在最大发射波长 520 nm 处呈黄-绿色荧光。

研究领域：Others

作用靶点：Fluorescent Dye

In Vitro: Protocol

1.Protein Preparetion
1) In order to obtain the best labeling effect, please prepare the protein (antibody) concentration as 2 mg/mL.
2) The pH value of protein solution shall be 8.5±0.5. If the pH is lower than 8.0, 1m sodium bicarbonate shall be used for adjustment.
3) If the protein concentration is lower than 2mg/ml, the labeling efficiency will be greatly reduced. In order to obtain the best labeling efficiency, it is recommended that the final protein concentration range is 2-10 mg/mL.
4) The protein must be in the buffer without primary amine (such as Tris or glycine) and ammonium ion, otherwise the labeling efficiency will be affected.
2.Dye Preparation
Add anhydrous DMSO into the vial of FITC to make a 10 mM stock solution. Mix well by pipetting or vortex.
3.Calculation of dye dosage
The amount of FITC required for reaction depends on the amount of protein to be labeled, and the optimal molar ratio of FITC to protein is about 10.
Example: assuming the required marker protein is 1 mL 2 mg/mL IgG (MW=150,000), use 1 mL DMSO dissolve 1 mg FITC, the required FITC volume is 40 μL.
4.Run conjugation reaction
1) A good volume of freshly prepared 10 mg/mL FITC is slowly added to 0.5 mL protein sample. In solution, gently shake to mix, then centrifuge briefly to collect the sample at the bottom of the reaction tube. Don't mix well to prevent protein samples from denaturation and inactivation.
2) The reaction tubules were placed in a dark place and incubated gently at room temperature for 60 minutes at intervals.For 10-15 minutes, gently reverse the reaction tubules several times to fully mix the two reactants and raise the bar efficiency.
5.Purify the conjugation
The following protocol is an example of dye-protein conjugate purification by using a Sephadex G-25 column.
1) Prepare Sephadex G-25 column according to the manufacture instruction.
2) Load the reaction mixture (From "Run conjugation reaction") to the top of the Sephadex G-25 column.
3) Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
4) Add more PBS (pH 7.2-7.4) to the desired sample to complete the column purification.
Combine the fractions that contain the desired dye-protein conjugate.

Note
1. FITC is sensitive to light and humidity. Immediately add FITC solution and discard the unused part.
2. Low concentrations of sodium azide (≤3 mM or 0.02%) or thiomersal (≤0.02 mM or 0.01%) did not significantly interfere with protein labeling; However, 20-50% glycerol will reduce labeling efficiency.
3. Avoid buffering with primary amines (e.g., Tris, glycine) or ammonium ions,It compete with labeled proteins.
4. This product is only for scientific research by professionals, and shall not be used in clinical diagnosis or treatment, food or medicine.
5. For your safety and health, please wear lab coat and disposable gloves.

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