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CY5

CAS No. : 146368-11-8

MCE 国际站：CY5

产品活性：CY5 是一种 CY 染料。CY 为花菁 (Cyanine) 的缩写，是由奇数个甲基单位连接的两个氮原子组成的化合物。菁类化合物具有波长长、吸收和发射可调、消光系数高、水溶性好、合成相对简单等特点。CY 系类染料常被用于蛋白，抗体以及小分子化合物的标记，对于蛋白抗体的标记，可以通过简单的混合反应来完成结合，以下我们介绍了蛋白抗体标记的标记方法，具有一定的参考意义。

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, 1 M sodium bicarbonate shall be used for adjustment.
3) If the protein concentration is lower than 2 mg/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 (Example for CY3-NHS ester)
Add anhydrous DMSO into the vial of CY3-NHS ester to make a 10 mM stock solution. Mix well by pipetting or vortex.
3.Calculation of dye dosage
The amount of CY3-NHS ester required for reaction depends on the amount of protein to be labeled, and the optimal molar ratio of CY3-NHS ester to protein is about 10.
Example: assuming the required marker protein is 500 μL 2 mg/mL IgG (MW=150,000), use 100 μL DMSO dissolve 1 mg CY3-NHS ester, the required CY3-NHS ester volume is 5.05 μL, and the detailed calculation process is as follows:
1) mmol (IgG) = mg/mL (IgG) ×mL (IgG) / MW (IgG) =2 mg/mL × 0.5 mL / 150,000 mg/mmol= 6.7×10-6 mmol
2) mmol (CY3-NHS ester) = mmol (IgG) × 10 = 6.7×10-6 mmol×10 = 6.7 × 10-5 mmol
3) μL (CY3-NHS ester) = mmol (CY3-NHS ester) ×MW (CY3-NHS ester) / mg/μL (CY3-NHS ester) = 6.7 ×10-5 mmol ×753.88 mg/mmol / 0.01 mg/μL = 5.05 μL (CY3-NHS ester)
4.Run conjugation reaction
1) A good volume of freshly prepared 10 mg/mL CY3-NHS ester 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'tmix 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.

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